DAP12 nucleic acids

ABSTRACT

The purification and isolation of various genes which encode mammalian cell surface polypeptides. Nucleic acids, proteins, antibodies, and other reagents useful in modulating development of cells, e.g., lymphoid and myeloid, are provided, along with methods for their use.

This filing is a divisional of U.S. Ser. No. 10/191,732, filed Jul. 8,2002, now U.S. Pat. No. 6,953,843, which is a divisional of U.S. Ser.No. 09/127,946, filed Jul. 31, 1998, now U.S. Pat. No. 6,416,973, whichclaims benefit of U.S. Provisional Patent Applications 60/089,168, filedJun. 12, 1998; 60/069,639, filed Dec. 15, 1997; 60/063,717, filed Oct.29, 1997; 60/069,692, filed Dec. 16, 1997; and 60/054,430, filed Aug. 1,1997; each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to various biological reagents which areuseful in modulating a mammalian cellular response, e.g., immunesignaling. More particularly, it is directed towards compositions andmethods useful in immune cell interactions, e.g., between B and T cells,NK, etc.

BACKGROUND OF THE INVENTION

The circulating component of the mammalian circulatory system comprisesvarious cell types, including red and white blood cells of the erythroidand myeloid cell lineages. See, e.g., Rapaport (1987) Introduction toHematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood:Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul(ed. 1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.

The activation of resting T cells is critical to most immune responsesand allows these cells to exert their regulatory or effectorcapabilities. See Paul (ed; 1993) Fundamental Immunology 3d ed., RavenPress, N.Y. Increased adhesion between T cells and antigen presentingcells (APC) or other forms of primary stimuli, e.g., immobilizedmonoclonal antibodies (mAb), can potentiate the T-cell receptor signals.T-cell activation and T cell expansion depends upon engagement of theT-cell receptor (TCR) and co-stimulatory signals provided by accessorycells. See, e.g., Jenkins and Johnson (1993) Curr. Opin. Immunol.5:361-367; Bierer and Hahn (1993) Semin. Immunol. 5:249-261; June, etal. (1990) Immunol. Today 11:211-216; and Jenkins (1994) Immunity1:443-446. A major, and well-studied, co-stimulatory interaction for Tcells involves either CD28 or CTLA-4 on T cells with either B7 or B70(Jenkins (1994) Immunity 1:443-446). Recent studies on CD28 deficientmice (Shahinian, et al. (1993) Science 261:609-612; Green, et al. (1994)Immunity 1:501-508) and CTLA-4 immunoglobulin expressing transgenic mice(Ronchese, et al. (1994) J. Exp. Med. 179:809-817) have revealeddeficiencies in some T-cell responses though these mice have normalprimary immune responses and normal CTL responses to lymphocyticchoriomeningitis virus and vesicular stomatitis virus. As a result, boththese studies conclude that other co-stimulatory molecules must besupporting T-cell function. However, identification of these moleculeswhich mediate distinct costimulatory signals has been difficult.

Moreover, similar negative and positive signaling occurs withlymphocytes (LIRs); natural killer cells (KIRs), and other cell types(ILT, and CD94). See, e.g., Moretta, et al. (1996) Ann. Rev. Immunol.14:619-648; Malissen (1996) Nature 384:518-519; Scharenberg and Kinet(1996) Cell 87:961-964; Colonna, et al. (1995) Science 268:405-408;Wagtmann, et al. (1995) Immunity 2:439-449; D'Andrea, et al. (1995) J.Immunol. 155:2306-2310; Samaridis and Colonna (1997) Eur. J. Immunol.27:660-665; Aramburu, et al. (1990) J. Immunol. 144:3238-3247; Aramburu,et al. (1991) J. Immunol. 147:714-721; and Rubio, et al. (1993) J.Immunol. 151:1312-1321.

The inability to modulate activation signals prevents control ofinappropriate developmental or physiological responses in the immunesystem. The present invention provides at least one alternativecostimulatory molecule, agonists and antagonists of which will be usefulin modulating a plethora of immune responses.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of particular genesinvolved in cell signaling. Various genes have been identified whichinteract with gene forms whose function was not understood. These arethe DNAX Accessory Protein, 12 kD (DAP12); the DNAX Accessory Protein,10 kD (DAP10); and another associated accessory protein, the MDL-1.

Particular embodiments of the invention include a substantially pure orrecombinant polypeptide exhibiting identity over a length of at leastabout 12 amino acids to the mature polypeptide from: SEQ ID NO: 2 or 6;SEQ ID NO: 8 or 10; or SEQ ID NO: 12 or 14. Preferably, the SEQ ID NO:is 2 or 6, and the polypeptide: is a mature natural sequence DAP12 fromTable 1; comprises an ITAM motif, or comprises a charged residue in atransmembrane domain; or the SEQ ID NO: is 8 or 10, and the polypeptide:is a mature natural sequence DAP10 from Table 2; comprises an ITIMmotif, or comprises a charged residue in a transmembrane domain; or theSEQ ID NO: is 12 or 14, and the polypeptide: is a mature naturalsequence MDL-1 of Table 3; or comprises a charged residue in atransmembrane domain. Other preferred embodiments include such apolypeptide which: comprises a plurality of the lengths; is a naturalallelic variant of DAP12; is a natural allelic variant of DAP10; is anatural allelic variant of MDL-1; has a length at least about 30 aminoacids; is a synthetic polypeptide; is attached to a solid substrate; isconjugated to another chemical moiety; is a 5-fold or less substitutionfrom natural sequence; or is a deletion or insertion variant from anatural sequence. Other preferred embodiments include a compositioncomprising: a sterile DAP12 polypeptide; the DAP12 polypeptide and acarrier, wherein the carrier is: an aqueous compound, including water,saline, and/or buffer; and/or formulated for oral, rectal, nasal,topical, or parenteral administration; or a sterile DAP10 polypeptide;or the DAP10 polypeptide and a carrier, wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration; or a sterile MDL-1 polypeptide; or the MDL-1 polypeptideand a carrier, wherein the carrier is: an aqueous compound, includingwater, saline, and/or buffer; and/or formulated for oral, rectal, nasal,topical, or parenteral administration.

A fusion protein is provided, comprising such a polypeptide and: adetection or purification tag, including a FLAG, His6, or immunoglobulinpeptide; bacterial β-galactosidase, trpE, Protein A, β-lactamase, alphaamylase, alcohol dehydrogenase, and yeast alpha mating factor; orsequence of another membrane protein.

Kits are provided comprising such a polypeptide and: a compartmentcomprising the polypeptide; and/or instructions for use or disposal ofreagents in the kit.

Binding compounds are also provided, comprising an antigen bindingportion from an antibody, which specifically binds to: a natural DAP12polypeptide, wherein the antibody: is raised against a maturepolypeptide of SEQ ID NO:2 or 6; is immunoselected; is a polyclonalantibody; binds to a denatured DAP12; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label; or a natural DAP10polypeptide, wherein the antibody: is raised against a maturepolypeptide of SEQ ID NO: 8 or 10; is immunoselected; is a polyclonalantibody; binds to a denatured DAP10; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label; or a natural MDL-1polypeptide, wherein the antibody: is raised against a maturepolypeptide of SEQ ID NO: 12 or 14; is immunoselected; is a polyclonalantibody; binds to a denatured MDL-1; exhibits a Kd to antigen of atleast 30 μM; is attached to a solid substrate, including a bead orplastic membrane; is in a sterile composition; or is detectably labeled,including a radioactive or fluorescent label. Various kits are provided,e.g., comprising the binding compound, and: a compartment comprising thebinding compound; and/or instructions for use or disposal of reagents inthe kit. Additional embodiments include a composition comprising: asterile binding compound, or the binding compound and a carrier, whereinthe carrier is: an aqueous compound, including water, saline, and/orbuffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding these polypeptides, wherein the nucleic acid encodes anantigenic peptide sequence of SEQ ID NO: 2, 6, 8, 10, 12, or 14.Preferred embodiments include such a nucleic acid, which encodes aplurality of antigenic peptide sequences of the table. Other nucleicacids include one which: is an expression vector; further comprises anorigin of replication; is from a natural source; comprises a detectablelabel; comprises synthetic nucleotide sequence; is less than 6 kb,preferably less than 3 kb; is from a mammal, including a primate orrodent; comprises a natural full length coding sequence; is ahybridization probe for a gene encoding DAP12, DAP10, or MDL-1; or is aPCR primer, PCR product, or mutagenesis primer

Other nucleic acids include ones which hybridize under stringent washconditions of at least 50° C., less than 400 mM salt, and 50% formamideto: SEQ ID NO: 1 or 5; SEQ ID NO: 7 or 9; or SEQ ID NO: 11 or 13. Theinvention provides a cell or tissue comprising such a recombinantnucleic acid, including where the cell is: a prokaryotic cell; aeukaryotic cell; a bacterial cell; a yeast cell; an insect cell; amammalian cell; a mouse cell; a primate cell; or a human cell. Certainkits include one comprising the nucleic acid, and: a compartmentcomprising the nucleic acid; a compartment further comprising a DAP12,DAP10, or MDL-1 polypeptide; and/or instructions for use or disposal ofreagents in the kit. Preferred nucleic acids include ones which: exhibitidentity over a stretch of at least about 30 nucleotides to a primateDAP12; exhibit identity over a stretch of at least about 30 nucleotidesto a primate DAP10; exhibit identity over a stretch of at least about 30nucleotides to a primate MDL-1; and/or further encode a KIR, ILT/MIR orCD94/NKG2C receptor. Preferred embodiments include those wherein: thewash conditions are at 60° C. and/or 200 mM salt; or the stretch is atleast 55 nucleotides.

The invention also provides methods of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting thecell with an agonist or antagonist of a DAP12, DAP10, or MDL-1. Also,methods are provided of screening for a compound which blocksinteraction of a DAP12 or DAP10 with a KIR, ILT/MIR, or CD94/NKG2Creceptor, comprising contacting the compound to the DAP12 or DAP10 inthe presence of the receptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Outline

I. General

II. Purified human DAP and MDL

-   -   A. physical properties    -   B. biological properties

III. Physical Variants

-   -   A. sequence variants, fragments    -   B. post-translational variants        -   1. glycosylation        -   2. others

IV. Functional Variants

-   -   A. analogs; fragments        -   1. agonists        -   2. antagonists    -   B. mimetics        -   1. protein        -   2. chemicals    -   C. polymorphic variants

V. Antibodies

-   -   A. polyclonal    -   B. monoclonal    -   C. fragments, binding compositions

VI. Nucleic Acids

-   -   A. natural isolates; methods    -   B. synthetic genes    -   C. methods to isolate

VII. Making DAP12; Mimetics

-   -   A. recombinant methods    -   B. synthetic methods    -   C. natural purification

VIII. Uses

-   -   A. diagnostic    -   B. therapeutic

IX. Kits

-   -   A. nucleic acid reagents    -   B. protein reagents    -   C. antibody reagents

X. Ligand or Counterreceptor

I. General

The present invention provides the amino acid sequences and DNAsequences of mammalian proteins which exhibit properties of accessorymolecules for cellular activation antigens. One protein is designatedDNAX Activation Protein, 12 kD (DAP12). The primate sequence describedherein was obtained from sequences identified from various databases.Similar sequences for proteins in other mammalian species should also beavailable, including rodent. The descriptions below are directed, forexemplary purposes, to the human DAP12 natural allele described, but arelikewise applicable to allelic and/or polymorphic variants, e.g., fromother individuals, as well as splicing variants, e.g., natural forms.

A second protein is designated DNAX Activation protein, 10 kD (DAP10),which exhibits many similar structural and biological features. A thirdprotein associates with the DAP12, and possibly with the DAP10, and isdesignated Myeloid DAP12 associated Lectin-1 (MDL-1).

These genes will allow isolation of other primate or mammalian genesencoding proteins related to them, further extending the family beyondthe specific embodiments described. The procedure is broadly set forthbelow.

The DNAX Activation Protein 12 kD (DAP12) is so named because of itsstructural features, and presumed function. Certain cell surfacereceptors lack intrinsic functionality, which hypothetically mayinteract with another protein partner, suggested to be a 12 kD protein.The mechanism of the signaling may involve an ITAM signal.

The DAP12 was identified from sequence databases based upon ahypothesized relationship to CD3 (see Olcese, et al. (1997) J. Immunol.158:5083-5086), the presence of an ITIM sequence (see Thomas (1995) J.Exp. Med. 181:1953-1956), certain size predictions (see Olcese; andTakase, et al. (1997) J. Immunol. 159:741-747, and other features. Inparticular, the transmembrane domain was hypothesized to contain acharged residue, which would allow a salt bridge with the correspondingtransmembrane segments of its presumed receptor partners, KIR (killercell inhibitory molecules) CD94 protein, and possibly other similarproteins. See Daeron, et al. (1995) Immunity 3:635-646.

In fact, many of the known KIR, MIR, ILT, and CD94/NKG2 receptormolecules may actually function with an accessory protein which is partof the functional receptor complex. See Olcese, et al. (1997) J.Immunol. 158:5083-5086; and Takase, et al. (1997) J. Immunol.159:741-747. Thus, the invention provides purified forms of thefunctional signaling receptors, e.g., the DAP12 and/or DAP10 with theother subunit. See, e.g., Daeron, et al. (1995) Immunity 3:635-646.Thus, a combination of DAP12 or DAP10 with another receptor forms afunctional complex on one cell which is a receptor complex for a counterreceptor or ligand for the complex.

The DAP10 was identified partly by its homology to the DAP12, and otherfeatures. In particular, in contrast to the DAP12, which exhibits anITAM activation motif, the DAP10 exhibits an ITIM inhibitory motif. TheMDL-1 was identified by its functional association with DAP12.

Moreover, the functional interaction between, e.g., DAP12 or DAP10, andits accessory receptor may allow use of the structural combination inreceptors which normally are not found in a truncated receptor form.Thus, the mechanism of signaling through such accessory proteins as theDAP12 and DAP10 allow for interesting engineering of other KIR-likereceptor complexes, e.g., with the KIR, MIR, ILT, and CD94 NKG2 typereceptors. Truncated forms of intact receptors may be constructed whichinteract with a DAP12 or DAP10 to form a functional signaling complex.

The primate and rodent forms exhibit significant sequence identity whenaligned. See, e.g., Tables 1, 2, and 3. Other genes exhibit much loweridentity over the entire mature coding region, though some exhibithigher identity in particular segments.

II. Purified DAP and MDL

Table 1 discloses both the nucleotide sequence (SEQ ID NO: 1 and 5) ofthe cDNA and the corresponding amino acid sequence for DAP12embodiments. The primate nucleotide sequence corresponds to SEQ ID NO:1; the amino acid sequence corresponds to SEQ ID NO: 2. The signalsequence appears to run from met (−26) to gln (−1) or ala1; the matureprotein should run from about ala1 (or gln2), the extracellular domainfrom about ala1 to pro14; the extracellular domain contains twocysteines at 7 and 9, which likely allow disulfide linkages toadditional homotypic or heterotypic accessory proteins; thetransmembrane region runs from about gly15 or val16 to about gly39; andan ITAM motif from tyr65 to leu79 (YxxL-6/8x-YxxL). The LVA03A EST wasidentified and used to extract other overlapping sequences. See alsoGenbank Human ESTs that are part of human DAP12; some, but not all,inclusive Genbank Accession #AA481924; H39980; W60940; N41026; R49793;W60864; W92376; H12338; T52100; AA480109; H12392; W74783; and T55959.

TABLE 1 Primate DAP12 cDNA identified from human cDNA library. SEQ IDNO: 1 and 2. Actual signal cleavage point may be slightly different fromthat indicated, e.g., may be between ala1 and gln2. ATG GGG GGA CTT GAACCC TGC AGC AGG CTC CTG CTC CTG CCT CTC CTG 48 Met Gly Gly Leu Glu ProCys Ser Arg Leu Leu Leu Leu Pro Leu Leu −26−25                 −20                 −15 CTG GCT GTA AGT GGT CTC CGTCCT GTC CAG GCC CAG GCC CAG AGC GAT 96 Leu Ala Val Ser Gly Leu Arg ProVal Gln Ala Gln Ala Gln Ser Asp−10                  −5                   1               5 TGC AGT TGCTCT ACG GTG AGC CCG GGC GTG CTG GCA GGG ATC GTG ATG 144 Cys Ser Cys SerThr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met             10                  15                  20 GGA GAC CTG GTGCTG ACA GTG CTC ATT GCC CTG GCC GTG TAC TTC CTG 192 Gly Asp Leu Val LeuThr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu         25                  30                  35 GGC CGG CTG GTC CCTCGG GGG CGA GGG GCT GCG GAG GCA GCG ACC CGG 240 Gly Arg Leu Val Pro ArgGly Arg Gly Ala Ala Glu Ala Ala Thr Arg     40                  45                  50 AAA CAG CGT ATC ACT GAGACC GAG TCG CCT TAT CAG GAG CTC CAG GGT 288 Lys Gln Arg Ile Thr Glu ThrGlu Ser Pro Tyr Gln Glu Leu Gln Gly 55                  60                  65                  70 CAG AGGTCG GAT GTC TAC AGC GAC CTC AAC ACA CAG AGG CCG TAT TAC 336 Gln Arg SerAsp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr                 75                  80                  85 AAA TGA 342Lys contig sequence with flanking untranslated regions (less reliable;possible sequence errors; SEQ ID NO: 3)CTTGCCTGGACGCTGCGCCACATCCCACCGGCCCTTACACTGTGGTGTCCAGCAGCATCCGGCTTCATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAgcGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAATGAGCCCGAATCATGACAGTCAGCAACATGATAcCTGGATCCAGCCATTCcTGAAGCCCAnCCTGCAcCTCATTCCAACTCCTACCGCGATACAGACCCACAGAGTGCCATCCCTGaGAGACCAGACCGCTCCCCAATACTCTCCTAAAATAAACATGAAGCACaAAAAAAAAAAAAAAAAAAACTCnGGGGGGGGGCCCGGTTAnCCAATTTGGnCCTAAAG Rodent DAP12 cDNAs, see mouse ESTs Genbanknumbers AA24315; W91184; AA098506; AA138406; W88159; and W41142. Aconsensus sequence, with filling in of holes, is well within the levelof skill in the art. See SEQ ID NO: 5 and 6. Signal cleavage point maybe to either side. ATG GGG GCT CTG GAG CCC TCC TGG TGC CTT CTG TTC CTTCCT GTC CTC 48 Met Gly Ala Leu Glu Pro Ser Trp Cys Leu Leu Phe Leu ProVal Leu −26 −25                 −20                 −15 CTG ACT GTG GGAGGA TTA AGT CCC GTA CAG GCC CAG AGT GAC ACT TTC 96 Leu Thr Val Gly GlyLeu Ser Pro Val Gln Ala Gln Ser Asp Thr Phe−10                  −5                   1               5 CCA AGA TGCGAC TGT TCT TCC GTG AGC CCT GGT GTA CTG GCT GGG ATT 144 Pro Arg Cys AspCys Ser Ser Val Ser Pro Gly Val Leu Ala Gly Ile             10                  15                  20 GTT CTG GGT GACTTG GTG TTG ACT CTG CTG ATT GCC CTG GCT GTG TAC 192 Val Leu Gly Asp LeuVal Leu Thr Leu Leu Ile Ala Leu Ala Val Tyr         25                  30                  35 TCT CTG GGC CGC CTGGTC TCC CGA GGT CAA GGG ACA GCG GAA GGG ACC 240 Ser Leu Gly Arg Leu ValSer Arg Gly Gln Gly Thr Ala Glu Gly Thr     40                  45                  50 CGG AAA CAA CAC ATT GCTGAG ACT GAG TCG CCT TAT CAG GAG CTT CAG 288 Arg Lys Gln His Ile Ala GluThr Glu Ser Pro Tyr Gln Glu Leu Gln 55                  60                  65                  70 GGT CAGAGA CCA GAA GTA TAC AGT GAC CTC AAC ACA CAG AGG CAA TAT 336 Gly Gln ArgPro Glu Val Tyr Ser Asp Leu Asn Thr Gln Arg Gln Tyr                 75                  80                  85 TAC AGA TGA345 Tyr Arg Alignment of primate and rodent DAP12 protein sequences (SEQID NO: 2 and 4). h MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ S--DCSCSTVSPGVLAGIVM m MGALEPSWCL LFLPVLLTVG GLSPVQAQSD TFPRCDCSSV SPGVLAGIVL hGDLVLTVLIA LAVYFLGRLV PRGRGAAEAA TRKQRITETE SPYQELQGQR m GDLVLTLLIALAVYSLGRLV SRGQGTAEG- TRKQHIAETE SPYQELQGQR h SDVYSDLNTQ RPYYK* mPEVYSDLNTQ RQYYR*

Table 2 discloses both the nucleotide sequence of the cDNA and thecorresponding amino acid sequence (SEQ ID NO: 7, 8, 9, and 10) of eachof the human and mouse DAP10 genes. The nucleotide sequence for humancorresponds to SEQ ID NO: 7; the amino acid sequence corresponds to SEQID NO: 8. The signal sequence appears to run from about met (−18) to ala(−1); the mature protein should run from about gln1, the extracellulardomain from about gln1 to pro30; the extracellular domain contains twocysteines at 21 and 24, which likely allow disulfide linkages toadditional homotypic or heterotypic accessory proteins; thetransmembrane region runs from about leu31 to val47, with acharacteristic charged residue corresponding to asp39; and aninteresting YxxM motif from tyr67 to met70, which is similar to thatseen in CD28, CTLA-4, and CD19. See Table 2.

Similarly, for the mouse DAP10, the signal sequence appears to run fromabout met (−18) to ser (−1); the mature protein should run from aboutgln1, the extracellular domain from about gln1 to pro 16; theextracellular domain contains two cysteines at 7 and 10, which likelyallow disulfide linkages to additional homotypic or heterotypicaccessory proteins; the transmembrane region runs from about leu17 toval33, with a characteristic charged residue corresponding to asp25; andan interesting YxxM motif from tyr54 to met57, which is similar to thatseen in CD28 and CTLA-4.

TABLE 2 Primate DAP10 cDNA identified from human cDNA library. See SEQID NO: 7 and 8. GTCGACCTGG ACTTCTCTGG ACCACAGTCC TCTGCCAGAC CCCTGCCAGACCCCAGTCCA 60 CC ATG ATC CAT CTG GGT CAC ATC CTC TTC CTG CTT TTG CTC CCAGTG 107    Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val   −18         −15                 −10                  −5 GCT GCA GCTCAG ACG ACT CCA GGA GAG AGA TCA TCA CTC CCT GCC TTT 155 Ala Ala Ala GlnThr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe              1               5                  10 TAC CCT GGC ACT TCAGGC TCT TGT TCC GGA TGT GGG TCC CTC TCT CTG 203 Tyr Pro Gly Thr Ser GlySer Cys Ser Gly Cys Gly Ser Leu Ser Leu     15                  20                  25 CCG CTC CTG GCA GGC CTCGTG GCT GCT GAT GCG GTG GCA TCG CTG CTC 251 Pro Leu Leu Ala Gly Leu ValAla Ala Asp Ala Val Ala Ser Leu Leu 30                  35                  40                  45 ATC GTGGGG GCG GTG TTC CTG TGC GCA CGC CCA CGC CGC AGC CCC GCC 299 Ile Val GlyAla Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala                 50                  55                  60 CAA GAT GGCAAA GTC TAC ATC AAC ATG CCA GGC AGG GGC TGACCCTCCT 348 Gln Asp Gly LysVal Tyr Ile Asn Met Pro Gly Arg Gly              65                  70GCAGCTTGGA CCTTTGACTT CTGACCCTCT CATCCTGGAT GGTGTGTGGT GCACAGGAAA 408CCCCGCCCCA ACTTTTGGAT TGTAATAAAA CATTTGAAAC ACA 451 Rodent DAP10 cDNAsequence from mouse library. See SEQ ID NO: 9 and 10. GTCACCATCGGGGTGACATC CGTCCTAGCT GCCTCTCTTC TCCTCTACTG TTCTGAGGAC 60 TTCCCTGGACCACAGTTTTG GCCAGATCCC TTCAGGTCCC AGCCCAGC ATG GAC CCC 117                                                     Met Asp Pro                                                     −18 CCA GGC TAC CTCCTG TTC CTG CTT CTG CTC CCA GTG GCT GCA AGT CAG 165 Pro Gly Tyr Leu LeuPhe Leu Leu Leu Leu Pro Val Ala Ala Ser Gln−15                 −10                  −5                   1 ACA TCGGCA GGT TCC TGC TCC GGA TGT GGG ACT CTG TCT CTG CCA CTC 213 Thr Ser AlaGly Ser Cys Ser Gly Cys Gly Thr Leu Ser Leu Pro Leu              5                  10                  15 CTG GCA GGC CTAGTG GCT GCA GAT GCG GTC ATG TCA CTC CTA ATT GTA 261 Leu Ala Gly Leu ValAla Ala Asp Ala Val Met Ser Leu Leu Ile Val         20                  25                  30 GGG GTG GTG TTT GTATGT ATG CGC CCA CAC GGC AGG CCT GCC CAA GAA 309 Gly Val Val Phe Val CysMet Arg Pro His Gly Arg Pro Ala Gln Glu     35                  40                  45 GAT GGT AGA GTC TAC ATCAAC ATG CCT GGC AGA GGC TGACCACGGC 355 Asp Gly Arg Val Tyr Ile Asn MetPro Gly Arg Gly  50                  55                  60 ACCTTCTGACCCGCTCATCC TGGATCCTGT GGGTTTGGGG TGCGTGGG 403 Alignment of primate androdent protein sequences (SEQ ID NO: 8 and 10). h: MIHLGHILFL LLLPVAAAQTTPGERSSLPA FYPGTSGSCS GCGSLSLPLL m: MDPPGYLLFL LLLPVAASQT S--------------AGSCS GCGTLSLPLL h: AGLVAADAVA SLLIVGAVFL CARPRRSPAQ -DGKVYINMPGRG* m: AGLVAADAVM SLLIVGVVFV CMRPHGRPAQ EDGRVYINMP GRG*

TABLE 3 Primate, e.g., human MDL-1 sequence (SEQ ID NO: 11 and 12).Because the designated methionine has no upstream termination codons, asexpected, it is conceivable that the protein actually has additionalupstream sequence. This methionione aligns with mouse sequence (seebelow). GGCTTAGCGT GGTCGCGGCC GAGGTGGCAA AAGGAGCATA TTCTCAGGAGACGGGGCCCC 60 TGCCTGCCAC ACCAAGCATT AGGCCACCAG GAAGACCCCC ATCTGCAAGCAAGCCTAGCC 120 TTCCAGGGAG AAAGAGGCCT CTGCAGCTCC TTCATC ATG AAC TGG CACATG ATC 174                                         Met Asn Trp His MetIle                                           1               5 ATC TCTGGG CTT ATT GTG GTA GTG CTT AAA GTT GTT GGA ATG ACC TTA 222 Ile Ser GlyLeu Ile Val Val Val Leu Lys Val Val Gly Met Thr Leu             10                  15                  20 TTT CTA CTT TATTTC CCA CAG ATT TTT AAC AAA AGT AAC GAT GGT TTC 270 Phe Leu Leu Tyr PhePro Gln Ile Phe Asn Lys Ser Asn Asp Gly Phe         25                  30                  35 ACC ACC ACC AGG AGCTAT GGA ACA GTC TCA CAG ATT TTT GGG AGC AGT 318 Thr Thr Thr Arg Ser TyrGly Thr Val Ser Gln Ile Phe Gly Ser Ser     40                  45                  50 TCC CCA AGT CCC AAC GGCTTC ATT ACC ACA AGG AGC TAT GGA ACA GTC 366 Ser Pro Ser Pro Asn Gly PheIle Thr Thr Arg Ser Tyr Gly Thr Val 55                  60                  65                  70 TGC CCCAAA GAC TGG GAA TTT TAT CAA GCA AGA TGT TTT TTC TTA TCC 414 Cys Pro LysAsp Trp Glu Phe Tyr Gln Ala Arg Cys Phe Phe Leu Ser                 75                  80                  85 ACT TCT GAATCA TCT TGG AAT GAA AGC AGG GAC TTT TGC AAA GGA AAA 462 Thr Ser Glu SerSer Trp Asn Glu Ser Arg Asp Phe Cys Lys Gly Lys             90                  95                 100 GGA TCC ACA TTGGCA ATT GTC AAC ACG CCA GAG AAA CTG TTT CTT CAG 510 Gly Ser Thr Leu AlaIle Val Asn Thr Pro Glu Lys Leu Phe Leu Gln        105                 110                 115 GAC ATA ACT GAT GCTGAG AAG TAT TTT ATT GGC TTA ATT TAC CAT CGT 558 Asp Ile Thr Asp Ala GluLys Tyr Phe Ile Gly Leu Ile Tyr His Arg    120                 125                 130 GAA GAG AAA AGG TGG CGTTGG ATC AAC AAC TCT GTG TTC AAT GGC AAT 606 Glu Glu Lys Arg Trp Arg TrpIle Asn Asn Ser Val Phe Asn Gly Asn135                 140                 145                 150 GTT ACCAAT CAG AAT CAG AAT TTC AAC TGT GCG ACC ATT GGC CTA ACA 654 Val Thr AsnGln Asn Gln Asn Phe Asn Cys Ala Thr Ile Gly Leu Thr                155                 160                 165 AAG ACC TTTGAT GCT GCA TCA TGT GAC ATC AGC TAC CGC AGG ATC TGT 702 Lys Thr Phe AspAla Ala Ser Cys Asp Ile Ser Tyr Arg Arg Ile Cys            170                 175                 180 GAG AAG AAT GCCAAA TGATCACAGT TCCCTGTGAC AAGAACTATA CTTGCAACTC 757 Glu Lys Asn Ala Lys        185 TTTTTGAATC CATAACAGGT CGTACTGGCC AATGATTACT TTTACTTACCTATCTGTACT 817 ACCAGTAGCG GTCCTTGCCC ATTTGGGAAA CTGAGCTTCT TTCTTCTGCACTGGGGGACT 877 GGATGCTAGC CATCTCCAGG AGACAGGATC AGTTTTACGG AAACAACTCAGTTAGTATAG 937 AGATGAGGTC CGCTTCTGTA GTACCTTCCT TCAAATAAAG AAATTTGGTACCTGCCCGG 996 Rodent, e.g., mouse, MDL-1 long form sequence (SEQ ID NO:13 and 14). A short form variant has been identified, which has adeletion of nucleotides 221-295. The short form variant characterizedalso possesses sequence differences: nucleotides 29-35 reads CAGAAGA;107-109 read AGA; 128-129 read AT; 820-826 read CATAGGT; lacks 859; and879-880 read CA. The initiation methionine has upstream terminationcodons suggesting it is the correct amino terminus. AGGACATTACCGAGCAGGAG CATACATTTC CAGAGCAAGG AGCCCTGCTC GCTGCACCGA 60 ATATCTTATCAAAAAGACTC CTATCTGTAT GCCAACCCAG ACTTCCCAGA AGAGATCAGA 120 TCCCTGATCCCCCATCATC ATG AAC TGG CAC ATG ATC ATC TCG GGG CTT ATC 172                     Met Asn Trp His Met Ile Ile Ser Gly Leu Ile                       1               5                  10 GTA GTA GTGATC AAA GTT GTT GGA ATG ACC TTT TTT CTG CTG TAT TTC 220 Val Val Val IleLys Val Val Gly Met Thr Phe Phe Leu Leu Tyr Phe             15                  20                  25 CCA CAG GTT TTTGGC AAA AGT AAT GAT GGC TTC GTC CCC ACG GAG AGC 268 Pro Gln Val Phe GlyLys Ser Asn Asp Gly Phe Val Pro Thr Glu Ser         30                  35                  40 TAC GGA ACC ACT AGTGTG CAG AAT GTC TCA CAG ATC TTT GGG AGA AAT 316 Tyr Gly Thr Thr Ser ValGln Asn Val Ser Gln Ile Phe Gly Arg Asn     45                  50                  55 GAC GAA AGT ACC ATG CCTACA AGG AGC TAT GGA ACA GTC TGT CCC AGA 364 Asp Glu Ser Thr Met Pro ThrArg Ser Tyr Gly Thr Val Cys Pro Arg 60                  65                  70                  75 AAC TGGGAT TTT CAC CAA GGA AAA TGC TTT TTC TTC TCC TTC TCC GAA 412 Asn Trp AspPhe His Gln Gly Lys Cys Phe Phe Phe Ser Phe Ser Glu                 80                  85                  90 TCA CCT TGGAAA GAC AGC ATG GAT TAT TGT GCA ACA CAA GGA TCC ACA 460 Ser Pro Trp LysAsp Ser Met Asp Tyr Cys Ala Thr Gln Gly Ser Thr             95                 100                 105 CTG GCA ATT GTCAAC ACT CCA GAG AAA CTG AAG TAT CTT CAG GAC ATA 508 Leu Ala Ile Val AsnThr Pro Glu Lys Leu Lys Tyr Leu Gln Asp Ile        110                 115                 120 GCT GGT ATT GAG AATTAC TTT ATT GGT TTG GTA CGT CAG CCT GGA GAG 556 Ala Gly Ile Glu Asn TyrPhe Ile Gly Leu Val Arg Gln Pro Gly Glu    125                 130                 135 AAA AAG TGG CGC TGG ATCAAC AAC TCT GTG TTC AAT GGC AAT GTT ACC 604 Lys Lys Trp Arg Trp Ile AsnAsn Ser Val Phe Asn Gly Asn Val Thr140                 145                 150                 155 AAT CAGGAC CAG AAC TTC GAC TGT GTC ACT ATA GGT CTG ACG AAG ACA 652 Asn Gln AspGln Asn Phe Asp Cys Val Thr Ile Gly Leu Thr Lys Thr                160                 165                 170 TAT GAT GCTGCA TCA TGT GAA GTC AGC TAT CGC TGG ATC TGC GAA ATG 700 Tyr Asp Ala AlaSer Cys Glu Val Ser Tyr Arg Trp Ile Cys Glu Met            175                 180                 185 AAT GCC AAATGATCATAGA TCTCTACAAG AGTGAATTTT TACAGAGCTA 749 Asn Ala Lys         190GCAAAGGAGA TTAGTTGTGA CTGAAACCAG CCCAGGAAAA TATAGAGCAT CAAAGACTGT 809GCCCATCTTC ATAGGTGGGA GTTCCCTATT GAATCCTCAA AGTCAATTTT GTTACTCCAC 869AAACATCTTA CCATAGTAAA ACTCCCT 896 Alignment of human MDL-1 (SEQ ID NO:12) and mouse MDL-1 long form (SEQ ID NO: 14). Of particular interestare a very short intracellular domain, corresponding to residues 1-2;with the transmembrane domain running from about 6 to 27 possessing acharged amino acid at about residue 16. Three putative N-linkedglycosylation sites correspond to residues 51, 146, and 153 of the mouselong form; the latter of which are conserved in the human sequence. Notethat the mouse long form, relative to the short form, appears to containa spacer segment of about 25 amino acids. hMDL-1MNWHMIISGLIVVVLKVVGMTLFLLYFPQIFNKSNDGFTTTRSYGT---- mMDL-1MNWHMIISGLIVVVIKVVGMTFFLLYFPQVFGKSNDGFVPTESYGTTSVQ**************.****** *******.*.******  * **** hMDL-1-VSQIFGSSSPSPNGFITTRSYGTVCPKDWEFYQARCFFLSTSESSWNES mMDL-1NVSQIFGRNDES---TMPTRSYGTVCPRNWDFHQGKCFFFSFSESPWKDS ******    *    . *********. *.* * .*** * *** * .* hMDL-1RDFCKGKGSTLAIVNTPEKL-FLQDITDAEKYFIGLIYHREEKRWRWINN mMDL-1MDYCATQGSTLAIVNTPEKLKYLQDIAGIENYFIGLVRQPGEKKWRWINN *.*  .************* .****.  * *****. .  **.****** hMDL-1SVFNGNVTNQNQNFNCATIGLTKTFDAASCDISYRRICEKNAK mMDL-1SVFNGNVTNQDQNFDCVTIGLTKTYDAASCEVSYRWICEMNAK********** *** * *******.*****..*** *** ***

As used herein, the term “human DAP12” shall refer, when used in aprotein context, to a protein having the primate amino acid sequenceshown in Table 1 of SEQ ID NO: 2. The present invention also encompassesproteins comprising a substantial fragment thereof, e.g., mutants andpolymorphic variants, along with a human derived polypeptide whichexhibits the same biological function or interacts with human DAP12specific binding components. These binding components typically bind toa human DAP12 with high affinity, e.g., at least about 100 nM, usuallybetter than about 30 nM, preferably better than about 10 nM, and morepreferably at better than about 3 nM. Homologous proteins are found inspecies other than humans, e.g., primates. While most of the descriptionbelow is directed to DAP12, similar methods and features may beanalogously applicable to the DAP10 and MDL-1 genes. Many limitationsdirected to DAP12 will correspond to terms in reference to DAP10 andMDL-1, though specific limitations relevant to one gene, e.g., a lengthlimitation, will not necessarily intended to apply to the others.

The term “polypeptide” as used herein includes a fragment or segment,and encompasses a stretch of amino acid residues of at least about 8amino acids, generally at least 10 amino acids, more generally at least12 amino acids, often at least 14 amino acids, more often at least 16amino acids, typically at least about 18 amino acids, more typically atleast about 20 amino acids, usually at least about 22 amino acids, moreusually at least about 24 amino acids, preferably at least about 26amino acids, more preferably at least about 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids, e.g., 33, 37, 41, 45, 49, 53, 57, 75, 100, 125, etc. In preferredembodiments, there will be a plurality of distinct, e.g.,nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima are provided,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12.

The term “binding composition” refers to molecules that bind withspecificity to DAP12, DAP10, or MDL-1, e.g., in an antibody-antigen typefashion. Other interactions include, e.g., receptor component-receptorcomponent, to form a receptor complex. Other members of the complex arelikely to be the KIR, LIR, MIR, ILT, and CD94 forms described above.Another interesting interaction includes such a receptor complex withits counter-receptor, which itself may be a single protein or complex.For instance, the receptor for the KIR-DAP12 complex will probably beMHC Class I. Such interactions will typically be a protein-proteininteraction, either covalent or non-covalent. The molecule may be apolymer, or chemical reagent. A functional analog may be a form withstructural modifications, or may be a wholly unrelated molecule whichhas a molecular shape which interacts with the appropriate surfacebinding determinants. The analogs may serve as agonists or antagonists,see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: ThePharmacological Bases of Therapeutics (8th ed.) Pergamon Press.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. and more usually greater than about 22° C. Fordiagnostic purposes, the temperature will usually be about roomtemperature or warmer, but less than the denaturation temperature ofcomponents in the assay. For therapeutic purposes, the temperature willusually be body temperature, typically about 37° C. for humans, thoughunder certain situations the temperature may be raised or lowered insitu or in vitro.

The electrolytes will usually approximate in situ physiologicalconditions, but may be modified to higher or lower ionic strength whereadvantageous. The actual ions may be modified to conform to standardbuffers used in physiological or analytical contexts.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions. Such proteins will be, e.g., soluble/short forms of theKIR, MIR, ILT, or CD94 proteins. Disruption of those complexes willtypically block the signal function.

The solvent will usually be a biologically compatible buffer, of a typeused for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS(3-([3-cholamidopropyl]-dimethylammonio)-1-propane sulfonate), or in alow enough detergent concentration to not disrupt the tertiary structureof the protein.

Solubility is reflected by sedimentation measured in Svedberg units,which are a measure of the sedimentation velocity of a molecule underparticular conditions. The determination of the sedimentation velocitywas classically performed in an analytical ultracentrifuge, but istypically now performed in a standard ultracentrifuge. See, Freifelder(1982) Physical Biochemistry (2d ed.), W.H. Freeman; and Cantor andSchimmel (1980) Biophysical Chemistry, parts 1-3, W.H. Freeman & Co.,San Francisco. As a crude determination, a sample containing aputatively soluble polypeptide is spun in a standard full sizedultracentrifuge at about 50K rpm for about 10 minutes, and solublemolecules will remain in the supernatant. A soluble particle orpolypeptide will typically be less than about 30 S, more typically lessthan about 15 S, usually less than about 10 S, more usually less thanabout 6 S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3 S.

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequences, e.g., of thehuman DAP12. It provides, e.g., 1-fold, 2-fold, 3-fold, 5-foldsubstitutions, preferably conservative. Such variants may be useful toproduce specific antibodies, and often will share many or all biologicalproperties.

Amino acid sequence identity is determined by optimizing residuematches. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Similar aminoacid sequences are intended to include natural allelic variations ineach respective protein sequence. Typical homologous proteins orpeptides will have from 85-100% identity (if gaps can be introduced), to90-100% identity (if conservative substitutions are included) with theamino acid sequence, e.g., of the human DAP12. Identity measures will beat least about 85%, generally at least about 87%, often at least about89%, typically at least about 91%, usually at least about 93%, moreusually at least about 95%, preferably at least about 97%, and morepreferably at least about 98%, and in particularly preferredembodiments, at least about 99% or more. See also Needleham, et al.(1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter One inTime Warps String Edits, and Macromolecules: The Theory and Practice ofSequence Comparison Addison-Wesley, Reading, Mass.; and softwarepackages from IntelliGenetics, Mountain View, Calif.; and the Universityof Wisconsin Genetics Computer Group, Madison, Wis.

The isolated human DAP and MDL DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of nucleotide stretches. These modifications will result innovel DNA sequences which encode useful antigens, their derivatives, orproteins having similar or antagonist activity. These modified sequencescan be used to produce mutant antigens or to enhance expression.Enhanced expression may involve gene amplification, increasedtranscription, increased translation, and other mechanisms. Such mutantDAP12 derivatives include predetermined or site-specific mutations ofthe respective protein or its fragments. “Mutant DAP12” encompasses apolypeptide otherwise sharing important features of the human DAP12 asset forth above, but having an amino acid sequence which differs fromthat of DAP12 as found in nature, whether by way of deletion,substitution, or insertion. In particular, “site specific mutant DAP12”is defined as having homology with an antigen of SEQ ID NO: 2, and assharing relevant biological activities with those antigens. Similarconcepts apply to different DAP12 proteins, particularly those found invarious other mammals. As stated before, it is emphasized thatdescriptions are generally meant to encompass additional DAP and MDLproteins, not limited solely to the primate embodiment specificallydiscussed.

Although site specific mutation sites are predetermined, mutants neednot be site specific. Human DAP12, DAP10, or MDL-1 mutagenesis can beconducted by making amino acid insertions or deletions. Substitutions,deletions, insertions, or any combinations may be generated to arrive ata final construct. Insertions include amino- or carboxy-terminalfusions. Random mutagenesis can be conducted at a target codon and theexpressed mutants can then be screened for the desired activity. Methodsfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis. See also Sambrook, et al. (1989) and Ausubel, et al. (1987and Supplements).

The mutations in the DNA normally should not place coding sequences outof reading frames and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structure such as loopsor hairpins.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of an immunoglobulin with, e.g., a DAP12 polypeptide, is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties derived from each source peptide. A similar concept appliesto heterologous nucleic acid sequences. Particularly interesting fusionswill be the DAP12 with its receptor partner, as discussed above. Bothprotein embodiments, and nucleic acids encoding both receptor complexcomponents will be valuable.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, partner-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.Thus, new chimeric polypeptides exhibiting new combinations ofspecificities will result from the functional linkage of partner-bindingspecificities and other functional domains.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

In certain situations, a DAP12 with multiple ITAM repeats, or an ITIMsubstitution, may be useful. Moreover, intact receptor functions may beachieved by splitting the long form of the transmembrane receptor intotwo separate subunits which interact as does the DAP12 with its partner.Thus, an intact long form receptor might be replaced with the pair of ashortened receptor with a DAP12. Nucleic acid constructs with thecombination may also be prepared. Likewise with DAP10, and ITIM repeats,or an ITAM substitution.

IV. Functional Variants

The blocking of physiological response to DAP12 or DAP10 antigens mayresult from the inhibition of binding of a partner to the DAP receptorcomplex, likely through competitive inhibition. Thus, in vitro assays ofthe present invention will often use isolated protein, membranes fromcells expressing a recombinant DAP12, soluble fragments comprisingpartner binding segments of these antigens, or fragments attached tosolid phase substrates. These assays will also allow for the diagnosticdetermination of the effects of either binding segment mutations andmodifications, or binding partner mutations and modifications.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the antigen or antigenfragments compete with a test compound for binding to the protein. Inthis manner, the antibodies can be used to detect the presence of apolypeptide which shares one or more binding sites of the antigen andcan also be used to occupy binding sites on the protein that mightotherwise be occupied by a binding partner. The invention alsocontemplates screening for compounds which interrupt the bridging of thecharged residues in the transmembrane segments between partners.

Additionally, neutralizing antibodies against the DAP or MDL and solublefragments of the DAP or MDL which contain a high affinity counterpartbinding site, can be used to inhibit binding function in tissues, e.g.,tissues experiencing abnormal physiology. Intracellular domaininteractions with other components will also be targets for drugscreening.

“Derivatives” of the DAP or MDL antigens include amino acid sequencemutants, glycosylation variants, and covalent or aggregate conjugateswith other chemical moieties. Covalent derivatives can be prepared bylinkage of functionalities to groups which are found in the DAP or MDLantigen amino acid side chains or at the N- or C-termini, by means whichare well known in the art. These derivatives can include, withoutlimitation, aliphatic esters or amides of the carboxyl terminus, or ofresidues containing carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g., lysine or arginine.Acyl groups are selected from the group of alkyl-moieties including C3to C18 normal alkyl, thereby forming alkanoyl aroyl species.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. While thereare no natural N-linked sites on the protein, there may be O-linkedsites, or variants with such sites may be produced. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., human glycosylation enzymes. Deglycosylation enzymesare also contemplated. Also embraced are versions of the same primaryamino acid sequence which have other minor modifications, includingphosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of, e.g., the DAP12antigens or fragments thereof with other proteins of polypeptides. Thesederivatives can be synthesized in recombinant culture such as N- orC-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred derivatization sites with cross-linking agents are at freeamino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between the DAP12 antigens and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different surface markers, resulting in, for instance, ahybrid protein exhibiting binding specificity of one or more markerproteins. Likewise, heterologous fusions may be constructed which wouldexhibit a combination of properties or activities of the derivativeproteins. Typical examples are fusions of a reporter polypeptide, e.g.,luciferase, with a segment or domain of an antigen, e.g., apartner-binding segment, so that the presence or location of a desiredpartner may be easily determined. See, e.g., Dull, et al., U.S. Pat. No.4,859,609, which is hereby incorporated herein by reference. Other genefusion partners include bacterial β-galactosidase, trpE, Protein A,β-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alphamating factor. See, e.g., Godowski, et al. (1988) Science 241:812-816.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity reagents.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, for example,in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded.) Vols. 1-3, Cold Spring Harbor Laboratory. Techniques for synthesisof polypeptides are described, for example, in Merrifield (1963) J.Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232: 341-347;and Atherton, et al. (1989) Solid Phase Peptide Synthesis: A PracticalApproach, IRL Press, Oxford.

This invention also contemplates the use of derivatives of the DAP12antigens other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, for example with cell membranes. Suchcovalent or aggregative derivatives are useful as immunogens, asreagents in immunoassays, or in purification methods such as foraffinity purification of binding partners. For example, a DAP12 antigencan be immobilized by covalent bonding to a solid support such ascyanogen bromide-activated Sepharose, by methods which are well known inthe art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-DAP12 antibodies or its binding partners. The DAP12 antigens canalso be labeled with a detectable group, for example radioiodinated ontoa tyrosine, e.g., incorporated into the natural sequence, by thechloramine T procedure, covalently bound to rare earth chelates, orconjugated to another fluorescent moiety for use in diagnostic assays.

A solubilized DAP or MDL antigen of this invention can be used as animmunogen for the production of antisera or antibodies specific for theantigen or many fragments thereof. The purified antigens can be used toscreen monoclonal antibodies or antigen-binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. In particular, the term “antibodies” also encompasses antigenbinding fragments of natural antibodies. The purified DAP or MDL canalso be used as a reagent to detect antibodies generated in response tothe presence of elevated levels of DAP, MDL, or cell fragmentscontaining the antigen, both of which may be diagnostic of an abnormalor specific physiological or disease condition. Additionally, DAPor MDLfragments may also serve as immunogens to produce the antibodies of thepresent invention, as described immediately below. For example, thisinvention contemplates antibodies raised against amino acid sequencesof, or encoded by nucleotide sequences, of SEQ ID NOs: 1-14 or fragmentsthereof. In particular, this invention contemplates antibodies havingbinding affinity to or being raised against specific fragments which arepredicted to lie outside of the lipid bilayer, either extracellular orintracellular domains. Additionally, various constructs may be producedfrom fusion of a membrane associating segment to the otherwiseextracellular exposed portion of the molecule. Other antigenic complexesmay be used, including complexes of the DAP or MDL with a receptorpartner.

The present invention contemplates the isolation of additional closelyrelated variants. It is highly likely that allelic variations exist indifferent individuals exhibiting, e.g., better than 90-97% identity tothe embodiment described herein.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of theantigens will be greatly accelerated by the isolation andcharacterization of distinct species counterparts of the antigens. Inparticular, the present invention provides useful probes for identifyingadditional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof DAP or MDL, e.g., either species types or cells which lackcorresponding antigens and exhibit negative background activity. Variouscell types, e.g., Jurkat, YT, or BAF3, transfected with CD94 or NKAT5may exhibit signaling when transfected also with DAP12. Expression oftransformed genes will allow isolation of antigenically pure cell lines,with defined or single specie variants. This approach will allow formore sensitive detection and discrimination of the physiological effectsof signaling. Subcellular fragments, e.g., cytoplasts or membranefragments, can be isolated and used.

Dissection of the critical structural elements which effect the variousdifferentiation functions provided by receptor binding is possible usingstandard techniques of modern molecular biology, particularly incomparing members of the related class. See, e.g., the homolog-scanningmutagenesis technique described in Cunningham, et al. (1989) Science243:1339-1336; and approaches used in O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO J.9:4381-4390.

In particular, receptor partner binding segments can be substitutedbetween species variants to determine what structural features areimportant in both binding affinity and specificity. An array ofdifferent, e.g., DAP12 variants, will be used to screen for partnersexhibiting combined properties of interaction with different speciesvariants.

Intracellular functions would probably involve segments of the antigenwhich are normally accessible to the cytosol. However, antigeninternalization may occur under certain circumstances, and interactionbetween intracellular components and the designated “extracellular”segments may occur. The specific segments of interaction of DAP12 withother intracellular components may be identified by mutagenesis ordirect biochemical means, e.g., cross-linking, affinity, or geneticmethods. Structural analysis by crystallographic or other physicalmethods will also be applicable. Further investigation of the mechanismof signal transduction will include study of associated components whichmay be isolatable by affinity methods.

Further study of the expression and control of DAP12 antigens will bepursued. The controlling elements associated with the antigens mayexhibit differential developmental, tissue specific, or other expressionpatterns. Upstream or downstream genetic regions, e.g., controlelements, are of interest.

Structural studies of the DAP12 antigens will lead to design of newvariants, particularly analogs exhibiting agonist or antagonistproperties. This can be combined with previously described screeningmethods to isolate variants exhibiting desired spectra of activities.

Expression in other cell types will often result in glycosylationdifferences in a particular antigen. Various species variants mayexhibit distinct functions based upon structural differences other thanamino acid sequence. Differential modifications may be responsible fordifferential function, and elucidation of the effects are now madepossible.

Although the foregoing description has focused primarily upon the humanDAP12, those of skill in the art will immediately recognize that theinvention encompasses other DAP12 antigens, e.g., primate and othermammalian species variants. In addition, the DAP10 gene exhibits manyfeatures similar to DAP12, and will be modifiable in similar fashion.There is evidence that the DAP12, DAP10, and MDL-1 may associate withone another, and may all be associated into one multiprotein complex incertain circumstances.

V. Antibodies

Antibodies can be raised to the various allelic or species variants ofDAP or MDL antigens and fragments thereof, both in their naturallyoccurring forms and in their recombinant forms. Additionally, antibodiescan be raised to DAP12 in either their active forms or in their inactiveforms, or native or denatured forms. Anti-idiotypic antibodies are alsocontemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of DAP or MDL can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective DAP or MDL, or screened for agonistic orantagonistic functional activity. These monoclonal antibodies willusually bind with at least a K_(D) of better than about 1 mM, moreusually better than about 300 μM, typically better than about 10 μM,more typically better than about 30 μM, preferably better than about 10μM, and more preferably better than about 3 μM, e.g., 1 μM, 300 nM, 100nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, etc.

The antibodies, including antigen binding fragments, of this inventioncan have significant diagnostic or therapeutic value. They can be potentantagonists that bind to DAP12, DAP10, of MDL-1, and/or inhibit partnerbinding or inhibit the ability to elicit a biological response. Theyalso can be useful as non-neutralizing antibodies and can be coupled totoxins or radionuclides so that when the antibody binds to the antigen,the cell itself is killed. Further, these antibodies can be conjugatedto drugs or other therapeutic agents, either directly or indirectly bymeans of a linker.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bindto the DAP or MDL without inhibiting partner binding and/or signaling.As neutralizing antibodies, they can be useful in competitive bindingassays. They will also be useful in detecting or quantifying DAP or MDLor its partners.

DAP12 fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. A DAP12 and its fragments may be fused or covalently linkedto a variety of immunogens, such as keyhole limpet hemocyanin, bovineserum albumin, tetanus toxoid, etc. See Microbiology, Hoeber MedicalDivision, Harper and Row, 1969; Landsteiner (1962) Specificity ofSerological Reactions, Dover Publications, New York, and Williams, etal. (1967) Methods in Immunology and Immunochemistry, Vol. 1, AcademicPress, New York, for descriptions of methods of preparing polyclonalantisera. A typical method involves hyperimmunization of an animal withan antigen. The blood of the animal is then collected shortly after therepeated immunizations and the gamma globulin is isolated.Alternatively, cells may be collected for producing hybridomas.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support, e.g., particles, such asagarose, SEPHADEX, or the like, where a cell lysate may be passedthrough the column, the column washed, followed by increasingconcentrations of a mild denaturant, whereby the purified DAP12 proteinwill be released.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against a DAP12, DAP10, or MDL-1 antigen will also beused to raise anti-idiotypic antibodies. These will be useful indetecting or diagnosing various immunological conditions related toexpression of the respective antigens.

A DAP12 protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ ID NO:2 or 6, is typically determined in an immunoassay. The immunoassaytypically uses a polyclonal antiserum which was raised, e.g., to aprotein of SEQ ID NO: 2 or 6. This antiserum is selected to have lowcrossreactivity against other CD3 family members, e.g., CD3 or FcεRγ,preferably from the same species, and any such crossreactivity isremoved by immunoabsorption prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2 or 6, or a combination thereof, is isolated as describedherein. For example, recombinant protein may be produced in a mammaliancell line. An appropriate host, e.g., an inbred strain of mice such asBalb/c, is immunized with the selected protein, typically using astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see Harlow and Lane, supra). Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Polyclonalsera are collected and titered against the immunogen protein in animmunoassay, e.g., a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity againstother CD3 family members, e.g., primate or rodent CD3, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably at least two CD3 familymembers are used in this determination in conjunction with either orsome of the primate or rodent DAP12. These DAP12 family members can beproduced as recombinant proteins and isolated using standard molecularbiology and protein chemistry techniques as described herein. Similartechniques may be applied to the DAP10 or MDL-1.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the proteins of SEQ ID NO:2 and/or 6 can be immobilized to a solid support. Proteins added to theassay compete with the binding of the antisera to the immobilizedantigen. The ability of the above proteins to compete with the bindingof the antisera to the immobilized protein is compared to the protein ofSEQ ID NO: 2 and/or 6. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the DAP12 like protein of SEQ ID NO: 2and/or 6). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired is less than twice the amount of the protein of the selectedprotein or proteins that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

VI. Nucleic Acids

The human DAP or MDL probe, or fragments thereof, will be used toidentify or isolate nucleic acids encoding homologous proteins fromother species, or other related proteins in the same or another species.Hybridization or PCR technology may be used.

This invention contemplates use of isolated DNA or fragments to encode,e.g., a biologically active corresponding DAP12 polypeptide. Inaddition, this invention covers isolated or recombinant DNA whichencodes a biologically active protein or polypeptide which is capable ofhybridizing under appropriate conditions with the DNA sequencesdescribed herein. Said biologically active protein or polypeptide can bean intact DAP12, or fragment, and have an amino acid sequence encoded bya nucleic acid of SEQ ID NO: 1 or 5. Further, this invention covers theuse of isolated or recombinant DNA, or fragments thereof, which encodesa protein which is homologous to a DAP12 or which was isolated usingcDNA encoding human DAP12 as a PCR or hybridization probe. The isolatedDNA can have the respective regulatory sequences in the 5′ and 3′flanks, e.g., promoters, enhancers, poly-A addition signals, and others.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and flanking genomic sequences from the originatingspecies. The invention embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity. Alternativelya mixture of purified sequences may be mixed, e.g., in a degenerate PCRapproach.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence. Alternatively, it can be a nucleic acid made bygenerating a sequence comprising fusion of two fragments which are notnaturally contiguous to each other, but is meant to exclude products ofnature, e.g., naturally occurring mutants. Thus, for example, productsmade by transforming cells with such an unnaturally occurring vector isencompassed, as are nucleic acids comprising sequence derived using asynthetic oligonucleotide process. Such is often done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing, e.g., a restriction or sequencerecognition site. Alternatively, it is performed to join togethernucleic acid segments of desired functions to generate a single geneticentity comprising a desired combination of functions not found in thecommonly available natural forms. Restriction enzyme recognition sitesare often the target of such artificial manipulations, but other sitespecific targets, e.g., promoters, DNA replication sites, regulationsequences, control sequences, or other useful features may beincorporated by design. A similar concept is intended for a recombinant,e.g., fusion, polypeptide. Specifically included are synthetic nucleicacids which, by genetic code redundancy, encode similar polypeptides tofragments of these antigens, and fusions of sequences from variousdifferent species variants.

A “fragment” in a nucleic acid context is a contiguous segment of atleast about 17 nucleotides, generally at least 20 nucleotides, moregenerally at least about 23 nucleotides, ordinarily at least about 26nucleotides, more ordinarily at least about 29 nucleotides, often atleast about 32 nucleotides, more often at least about 35 nucleotides,typically at least about 38 nucleotides, more typically at least about41 nucleotides, usually at least about 44 nucleotides, more usually atleast about 47 nucleotides, preferably at least about 50 nucleotides,more preferably at least about 53 nucleotides, and in particularlypreferred embodiments will be at least about 56 or more nucleotides,e.g., 60, 75, 100, 150, 200, 250, 300, etc.

A DNA which codes for, e.g., a DAP12 protein, will be particularlyuseful to identify genes, mRNA, and cDNA species which code for relatedor homologous antigens, as well as DNAs which code for homologousproteins from different species. Various DAP12 proteins should besimilar in sequence and are encompassed herein. However, even proteinsthat have a more distant evolutionary relationship to the DAP12 canreadily be isolated using these sequences if they exhibit sufficientsimilarity. Primate DAP12, DAP10, and MDL-1 proteins are of particularinterest.

This invention further encompasses recombinant DNA molecules andfragments having a DNA sequence identical to or highly homologous to theisolated DNAs set forth herein. In particular, the sequences will oftenbe operably linked to DNA segments which control transcription,translation, and DNA replication. Alternatively, recombinant clonesderived from the genomic sequences, e.g., containing introns, will beuseful for transgenic studies, including, e.g., transgenic cells andorganisms, and for gene therapy. See, e.g., Goodnow (1992) “TransgenicAnimals” in Roitt (ed.) Encyclopedia of Immunology Academic Press, SanDiego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al.(1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson(1987)(ed.) Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach IRL Press, Oxford; and Rosenberg (1992) J. Clinical Oncology10:180-199. Operable association of heterologous promoters with naturalgene sequences is also provided, as are vectors encoding, e.g., theDAP12 with a receptor partner.

Homologous nucleic acid sequences, when compared, exhibit significantsequence similarity. The standards for homology in nucleic acids areeither measures for homology generally used in the art by sequencecomparison or based upon hybridization conditions. The hybridizationconditions are described in greater detail below.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

Substantial identity in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least about 56%, more generally at least about59%, ordinarily at least about 62%, more ordinarily at least about 65%,often at least about 68%, more often at least about 71%, typically atleast about 74%, more typically at least about 77%, usually at leastabout 80%, more usually at least about 85%, preferably at least about90%, more preferably at least about 95 to 98% or more, and in particularembodiments, as high at about 99% or more of the nucleotides.Alternatively, substantial identity exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a sequence of SEQ ID NO: 1 or 5. Typically,selective hybridization will occur when there is at least about 55%homology over a stretch of at least about 14 nucleotides, preferably atleast about 65%, more preferably at least about 75%, and most preferablyat least about 90%. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. Thelength of homology comparison, as described, may be over longerstretches, and in certain embodiments will be over a stretch of at leastabout 17 nucleotides, usually at least about 20 nucleotides, moreusually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 40 nucleotides, preferably atleast about 50 nucleotides, and more preferably at least about 75 to 100or more nucleotides, e.g., 125, 150, 200, 250, 300, etc.

Stringent conditions, in referring to identity in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., more usually in excessof about 37° C., typically in excess of about 45° C., more typically inexcess of about 55° C., preferably in excess of about 65° C., and morepreferably in excess of about 70° C. Stringent salt conditions willordinarily be less than about 500 mM, usually less than about 350 mM,more usually less than about 200 mM, typically less than about 150 mM,preferably less than about 100 mM, and more preferably less than about50 mM. However, the combination of parameters is much more importantthan the measure of any single parameter. See, e.g., Wetmur and Davidson(1968) J. Mol. Biol. 31:349-370. Hybridization under stringentconditions should give a background of at least 2-fold over background,preferably at least 3-5 or more.

DAP or MDL from other human subjects can be cloned and isolated byhybridization or PCR. Alternatively, preparation of an antibodypreparation which exhibits less allelic specificity may be useful inexpression cloning approaches. Allelic variants may be characterizedusing, e.g., a combination of redundant PCR and sequence analysis, e.g.,using defined primers, thereby providing information on allelicvariation in a human population.

VII. Making DAP or MDL; Mimetics

DNA which encodes the DAP or MDL antigen or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or byscreening genomic libraries prepared from a wide variety of cell linesor tissue samples.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length antigen or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies. Each antigen or its fragments can beexpressed in host cells that are transformed or transfected withappropriate expression vectors. These molecules can be substantiallypurified to be free of protein or cellular contaminants, e.g., thosederived from the recombinant host, and therefore are particularly usefulin pharmaceutical compositions when combined with a pharmaceuticallyacceptable carrier and/or diluent. The antigen, or portions thereof, maybe expressed as fusions with other proteins.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired antigen gene or its fragments, usually operablylinked to suitable genetic control elements that are recognized in asuitable host cell. These control elements are capable of effectingexpression within a suitable host. The specific type of control elementsnecessary to effect expression will depend upon the eventual host cellused. Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell.

The vectors of this invention contain DNA which encodes, e.g., a humanDAP12 antigen, or a fragment thereof encoding a biologically activepolypeptide. The DNA can be under the control of a viral promoter andcan encode a selection marker. This invention further contemplates useof such expression vectors which are capable of expressing eukaryoticcDNA coding for a primate DAP12 antigen in a prokaryotic or eukaryotichost, where the vector is compatible with the host and where theeukaryotic cDNA coding for the antigen is inserted into the vector suchthat growth of the host containing the vector expresses the cDNA inquestion. Usually, expression vectors are designed for stablereplication in their host cells or for amplification to greatly increasethe total number of copies of the desirable gene per cell. It is notalways necessary to require that an expression vector replicate in ahost cell, e.g., it is possible to effect transient expression of theantigen or its fragments in various hosts using vectors that do notcontain a replication origin that is recognized by the host cell. It isalso possible to use vectors that cause integration of the human DAP12gene or its fragments into the host DNA by recombination.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but all other forms of vectors which servean equivalent function and which are, or become, known in the art aresuitable for use herein. See, e.g., Pouwels, et al. (1985 andSupplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., andRodriquez, et al. (1988)(eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, Mass.

Transformed cells are cells, preferably mammalian, that have beentransformed or transfected with human DAP12 vectors constructed usingrecombinant DNA techniques. Transformed host cells usually express theantigen or its fragments, but for purposes of cloning, amplifying, andmanipulating its DNA, do not need to express the protein. This inventionfurther contemplates culturing transformed cells in a nutrient medium,thus permitting the protein to accumulate in the culture. The proteincan be recovered, either from the culture or from the culture medium.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression.

Suitable host cells include, e.g., prokaryotes, lower eukaryotes, andhigher eukaryotes. Prokaryotes include both gram negative and grampositive organisms, e.g., E. coli and B. subtilis. Lower eukaryotesinclude yeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express, e.g., thehuman DAP12 antigens or its fragments include, but are not limited to,such vectors as those containing the lac promoter (pUC-series); trppromoter (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pRpromoters (pOTS); or hybrid promoters such as ptac (pDR540). SeeBrosius, et al. (1988) “Expression Vectors Employing Lambda-, trp-,lac-, and Ipp-derived Promoters”, in Rodriguez and Denhardt (eds.)Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Buttersworth, Boston, Chapter 10, pp. 205-236.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith, e.g., human DAP12 antigen sequence containing vectors. Forpurposes of this invention, the most common lower eukaryotic host is thebaker's yeast, Saccharomyces cerevisiae. It will be used to genericallyrepresent lower eukaryotes although a number of other strains andspecies are also available. Yeast vectors typically consist of areplication origin (unless of the integrating type), a selection gene, apromoter, DNA encoding the desired protein or its fragments, andsequences for translation termination, polyadenylation, andtranscription termination. Suitable expression vectors for yeast includesuch constitutive promoters as 3-phosphoglycerate kinase and variousother glycolytic enzyme gene promoters or such inducible promoters asthe alcohol dehydrogenase 2 promoter or metallothionine promoter.Suitable vectors include derivatives of the following types:self-replicating low copy number (such as the YRp-series),self-replicating high copy number (such as the YEp-series); integratingtypes (such as the YIp-series), or mini-chromosomes (such as theYCp-series).

Higher eukaryotic tissue culture cells are the preferred host cells forexpression of the functionally active human DAP or MDL antigen protein.In principle, many higher eukaryotic tissue culture cell lines areworkable, e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred, in that the processing, both cotranslationally andposttranslationally. Transformation or transfection and propagation ofsuch cells has become a routine procedure. Examples of useful cell linesinclude HeLa cells, Chinese hamster ovary (CHO) cell lines, baby ratkidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey(COS) cell lines. Expression vectors for such cell lines usually includean origin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pcDNA1; pCD, see Okayama, et al. (1985) Mol.Cell. Biol. 5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

It will often be desired to express a human DAP or MDL antigenpolypeptide in a system which provides a specific or definedglycosylation pattern. In this case, the usual pattern will be thatprovided naturally by the expression system. However, the pattern willbe modifiable by exposing the polypeptide, e.g., an unglycosylated form,to appropriate glycosylating proteins introduced into a heterologousexpression system. For example, the DAP12 antigen gene may beco-transformed with one or more genes encoding mammalian or otherglycosylating enzymes. Using this approach, certain mammalianglycosylation patterns will be achievable or approximated in prokaryoteor other cells.

The DAP antigens might also be produced in a form which is phosphatidylinositol (PI) linked, but can be removed from membranes by treatmentwith a phosphatidyl inositol cleaving enzyme, e.g., phosphatidylinositol phospholipase-C. This releases the antigen in a biologicallyactive form, and allows purification by standard procedures of proteinchemistry. See, e.g., Low (1989) Biochim. Biophys. Acta 988:427-454;Tse, et al. (1985) Science 230:1003-1008; and Brunner, et al. (1991) J.Cell Biol. 114:1275-1283. Alternatively, purification segments may beengineered into the sequence, e.g., at the N-terminus or C-terminus, toassist in the purification or detection of the protein product. Means toremove such segments may also be engineered, e.g., protease cleavagesites.

Now that the entire sequences are known, the primate DAP or MDLantigens, fragments, or derivatives thereof can be prepared byconventional processes for synthesizing peptides. These includeprocesses such as are described in Stewart and Young (1984) Solid PhasePeptide Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky andBodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, NewYork; and Bodanszky (1984) The Principles of Peptide Synthesis,Springer-Verlag, New York. For example, an azide process, an acidchloride process, an acid anhydride process, a mixed anhydride process,an active ester process (for example, p-nitrophenyl ester,N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazoleprocess, an oxidative-reductive process, or a dicyclohexylcarbodiimide(DCCD)/additive process can be used. Solid phase and solution phasesyntheses are both applicable to the foregoing processes.

The human DAP or MDL antigens, fragments, or derivatives are suitablyprepared in accordance with the above processes as typically employed inpeptide synthesis, generally either by a so-called stepwise processwhich comprises condensing an amino acid to the terminal amino acid, oneby one in sequence, or by coupling peptide fragments to the terminalamino acid. Amino groups that are not being used in the couplingreaction must be protected to prevent coupling at an incorrect location.

If a solid phase synthesis is adopted, the C-terminal amino acid isbound to an insoluble carrier or support through its carboxyl group. Theinsoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins,tert-alkyloxycarbonyl-hydrazidated resins, and the like.

An amino group-protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid-phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149-2156.

The prepared antigen and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, for example,by extraction, precipitation, electrophoresis and various forms ofchromatography, and the like. The human DAP12 antigens of this inventioncan be obtained in varying degrees of purity depending upon its desireduse. Purification can be accomplished by use of the protein purificationtechniques disclosed herein or by the use of the antibodies hereindescribed, e.g., in immunoabsorbent affinity chromatography. Thisimmunoabsorbent affinity chromatography is carried out, e.g., by firstlinking the antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of cells, lysates of other cellsexpressing, e.g., the DAP12 antigens, or lysates or supernatants ofcells producing the DAP12 antigens as a result of DNA techniques, seebelow.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental or physiological abnormalities, orbelow in the description of kits for diagnosis.

Many of the receptors important in the activation of leukocytes(including the T cell antigen receptor, and immunoglobulin and Fcreceptors) lack intrinsic signaling properties, but transmit theirsignals by coupling non-covalently with other membrane proteins thatcontain immunoreceptor tyrosine-based activation motifs (ITAM, YxxL-6 to8 amino acid spacer-YxxL) in their cytoplasmic domains. For example, theT cell antigen receptor is associated with the CD3 gamma, delta,epsilon, and zeta proteins that contain ITAM sequences. Similarly,surface immunoglobulin on B cells is associated with CD79A and CD79Bthat contain ITAM and are required for signal transduction. The Fcreceptors for IgG (CD16) on NK cells associates with CD3 zeta or the IgEFc receptor-gamma subunit (both containing ITAM) and the high affinityIgE receptor on mast cells associated with the IgE Fc receptor-gammasubunit. Therefore, associated proteins containing ITAM represent ageneral strategy in the assembly of activating receptors on leukocytes.

Recently, several new families of leukocyte receptors have beenidentified that are structurally diverse. Certain isoforms of the KIR,ILT/MIR, Ly49, and CD94/NKG2 family of receptors have been implicated inpositive signaling; however, these molecules (e.g. KIR-NKAT5, KIR-c139,ILT1, gp91/PIR, and CD94) lack sequences in their cytoplasmic domainsthat would be consistent with positive signaling capability.

Given that T cell antigen receptors, immunoglobulin receptors, and Fcreceptors all achieve signaling function by association with anothersmall subunit containing ITAM, it is likely that these other leukocytereceptors might use a similar strategy.

Therefore, available sequence databases were searched with proteinsequences of human and mouse CD3 gamma, delta, epsilon, and zeta, andIgE Fc receptor-gamma chain. An EST designated LVA03A was identifiedthat encodes a putative membrane protein of ˜12 kd with an acidicresidue (D) in the transmembrane segment and a perfect ITAM sequence inthe cytoplasmic domain. Cysteine residues in the short extracellulardomain suggest the molecule might be expressed as a disulfide-bondeddimer. Distribution studies indicate the gene is transcribed inmacrophages, dendritic cells, some T cells, and NK cells. This proteinhas been designated DNAX Activating Protein 12 (DAP12). An analogousgene was also identified, designated DAP10, which possesses ITIM motifs.

Receptors containing ITAM have all been important in inducing leukocytefunction (e.g., T cell antigen receptor, immunoglobulin receptor, Fcreceptor). Therefore, it is probably that DAP12 will have an importantrole in signal transduction in leukocytes. Agonists and antagonists ofDAP12 should provide useful in either potentiating or inhibiting immuneresponses (i.e., proliferation, cytokine production, inducing apoptosis,or triggering cell-mediated cytotoxicity), respectively.

Receptors containing the YxxM motif have been identified as important incertain signaling molecules, e.g., CD28, CTLA-4, and CD19. Therefore, itis probably that DAP10 will have an important role in signaltransduction. Agonists and antagonists of DAP10 should provide useful ineither potentiating or inhibiting immune responses (i.e., proliferation,cytokine production, inducing apoptosis, or triggering cell-mediatedcytotoxicity), respectively.

It is anticipated that DAP12 may non-covalently associate with severaldifferent membrane receptors, for example, but not necessarily limitedto T cell antigen receptor, the pre-T cell antigen receptor, theimmunoglobulin receptor, Fc receptors, the KIR family of receptors, theILT/MIR family of receptors, the LAIR family of receptors, the gp91/PIRfamily of receptors, the Ly49 family of receptors (specifically Ly49Dand Ly49H), and the CD94/NKG2 family of receptors. Among these is theMDL-1. Therefore, reagents to affect DAP12 interaction with saidreceptors may either enhance or suppress the function of these moleculesfor therapeutic intervention (i.e., augment immunity for vaccination orimmunodeficiency diseases or suppress immune responses in the case ofautoimmune diseases or transplantation). Combinations of DAP with anyone of these receptors will be useful, e.g., for drug screening forinterrupters of the interaction and subsequent signaling, as willantibodies to the structural complexes arising form their interaction.

The DAP12 may be playing a role in Beta2 like integrin signaling. It isclear that Beta2 integrin can transmit a P Tyr kinase dependent signalinvolving Syk. In Syk knockouts, Beta2 does not signal. The pathway alsoprobably involves FcγR (in Monocytes/Macrophages and B cells) as anegative regulator. However, there is no known way for Syk to associatewith Beta2 integrins as they have no ITAM containing sequences in therecytoplasmic domains. Moreover, there is no evidence that the known ITAMcontaining proteins can associate with Beta2. Thus, DAP12 would be aprime candidate or prototype for one that would associate with Beta2.

This invention also provides reagents with significant therapeuticvalue. The human DAP12 or DAP10 (naturally occurring or recombinant),fragments thereof and antibodies thereto, along with compoundsidentified as having binding affinity to primate DAP, should be usefulin the treatment of conditions associated with abnormal B cell response,including abnormal proliferation, e.g., cancerous conditions, ordegenerative conditions. Abnormal proliferation, regeneration,degeneration, and atrophy may be modulated by appropriate therapeutictreatment using the compositions provided herein. For example, a diseaseor disorder associated with abnormal expression or abnormal triggeringof DAP12 should be a likely target for an agonist or antagonist of theantigen. DAP12 likely plays a role in activation or regulation of immunecells, which affect immunological responses, e.g., autoimmune disordersor allergic responses.

In addition, the DAP:DAP binding partner interaction may be involved inT, NK, DC, or monocyte cell interactions that permit the activation,proliferation, and/or differentiation interacting cells. If so,treatment may result from interference with the DAP:DAP binding partnersignal transduction, particularly potentiating or inhibiting immuneresponses such as proliferation, cytokine production, inducingapoptosis, or triggering cell-mediated cytotoxicity. Blocking of thesignal may be effected, e.g., by soluble DAP or antibodies to DAP, ordrugs which disrupt the functional interaction of the DAP with itsreceptor complex partner.

Other abnormal developmental conditions are known in each of the celltypes shown to possess DAP12 or DAP10 mRNA by Northern blot analysis,e.g., lymphocytes, NK, monocytes, and dendritic cells. See Berkow (ed.)The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.;and Thorn, et al. Harrison's Principles of Internal Medicine,McGraw-Hill, N.Y. For example, therapeutic immunosuppression may beachieved by blocking T lymphocyte and B lymphocyte interaction throughthis molecule. It will represent an important therapy for controllingautoimmune diseases and graft rejection during transplantation. Theblockage may be effected with blocking binding compositions, e.g.,neutralizing antibodies.

Recombinant DAP or DAP antibodies can be purified and then administeredto a patient. These reagents can be combined for therapeutic use withadditional active ingredients, e.g., in conventional pharmaceuticallyacceptable carriers or diluents, e.g., immunogenic adjuvants, along withphysiologically innocuous stabilizers and excipients. Thesecombinations, and compositions provided, can be sterile filtered andplaced into dosage forms as by lyophilization in dosage vials or storagein stabilized aqueous preparations. This invention also contemplates useof antibodies or binding fragments thereof which are not complementbinding.

Drug screening using DAP or fragments thereof can be performed toidentify compounds having binding affinity to a DAP, including isolationof associated components. Subsequent biological assays can then beutilized to determine whether the compound has intrinsic stimulatingactivity and is therefore a blocker or antagonist in that it blockssignaling. Likewise, a compound having intrinsic stimulating activitycan activate the antigen and is thus an agonist in that it simulates theactivity of a DAP. This invention further contemplates the therapeuticuse of antibodies to DAP as antagonists. This approach should beparticularly useful with other DAP or MDL species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

Human DAP or MDL, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for topical, oral,rectal, nasal, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form, in sterile forms, or maybe prepared by many methods well known in the art of pharmacy. See,e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; andRemington's Pharmaceutical Sciences, 17th ed. (1990), Mack PublishingCo., Easton, Pa. The therapy of this invention may be combined with orused in association with other agents.

Both the naturally occurring and the recombinant forms of the DAP or MDLantigens of this invention are particularly useful in kits and assaymethods which are capable of screening compounds for binding activity tothe proteins. Several methods of automating assays have been developedin recent years so as to permit screening of tens of thousands ofcompounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, which describes means for testing of binding affinity by aplurality of defined polymers synthesized on a solid substrate. Thedevelopment of suitable assays can be greatly facilitated by theavailability of large amounts of purified, soluble DAP or MDL asprovided by this invention.

For example, antagonists can normally be found once a DAP or MDL hasbeen structurally defined. Testing of potential antagonists is nowpossible upon the development of highly automated assay methods using apurified DAP or MDL. In particular, new agonists and antagonists will bediscovered by using screening techniques made available herein. Ofparticular importance are compounds found to have a combined bindingaffinity for multiple DAP12, DAP10, or MDL-1 proteins, e.g., compoundswhich can serve as antagonists for allelic variants of DAP or MDL.

Moreover, since the signaling through the DAP:DAP binding partner mayfunction in combination with other signals, combination therapy withsuch pathways will also be considered. Thus, antagonism of multiplesignal pathways, or stimulation with multiple pathways may be useful.Moreover, with the association of the DAP12 with MDL-1, and possiblyalso with DAP10, various combinations of the described genes may beimportant.

This invention is particularly useful for screening compounds by usingthe recombinant antigens in any of a variety of drug screeningtechniques. The advantages of using a recombinant protein in screeningfor specific compounds include: (a) improved renewable source of theDAP12 from a specific source; (b) potentially greater number of antigenmolecules per cell giving better signal to noise ratio in assays; and(c) species variant specificity (theoretically giving greater biologicaland disease specificity).

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing the DAP and/or MDL. Cells may be isolated which express a DAPin isolation from others, or in combination with its receptor complexpartner. Such cells, either in viable or fixed form, can be used forstandard antigen/partner binding assays. See also, Parce, et al. (1989)Science 246:243-247; and Owicki, et al. (1990) Proc. Nat'l Acad. Sci.USA 87:4007-4011, which describe sensitive methods to detect cellularresponses. Competitive assays are particularly useful, where the cells(source of DAP) are contacted and incubated with a labeled compoundhaving known binding affinity to the antigen, and a test compound whosebinding affinity to the DAP is being measured. The bound compound andfree compound are then separated to assess the degree of binding. Theamount of test compound bound is inversely proportional to the amount oflabeled compound binding measured. Many techniques can be used toseparate bound from free compound to assess the degree of binding. Thisseparation step could typically involve a procedure such as adhesion tofilters followed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes. Viable cells could also be used toscreen for the effects of drugs on DAP mediated functions, e.g., secondmessenger levels, i.e., Ca⁺⁺; cell proliferation; inositol phosphatepool changes; and others. Some detection methods allow for eliminationof a separation step, e.g., a proximity sensitive detection system.Calcium sensitive dyes will be useful for detecting Ca⁺⁺ levels, with afluorimeter or a fluorescence cell sorting apparatus.

Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of the human DAP or MDL. Thesecells are stably transformed with DNA vectors directing the expressionof human DAP or MDL antigen. Essentially, the membranes would beprepared from the cells and used in a receptor complex binding assaysuch as the competitive assay set forth above.

Still another approach is to use solubilized, unpurified or solubilized,purified DAP from transformed eukaryotic or prokaryotic host cells. Thisallows for a “molecular” binding assay with the advantages of increasedspecificity, the ability to automate, and high drug test throughput.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto human DAP or MDL and is described in detail in Geysen, EuropeanPatent Application 84/03564, published on Sep. 13, 1984. First, largenumbers of different small peptide test compounds are synthesized on asolid substrate, e.g., plastic pins or some other appropriate surface,see Fodor, et al. (1991). Then all the pins are reacted withsolubilized, unpurified or solubilized, purified DAP, and washed. Thenext step involves detecting bound DAP.

Rational drug design may also be based upon structural studies of themolecular shapes of the DAP or MDL and other effectors. Effectors may beother proteins which mediate other functions in response to receptorcomplex binding, or other proteins which normally interact with theantigen. One means for determining which sites interact with specificother proteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions.For a detailed description of protein structural determination, see,e.g., Blundell and Johnson (1976) Protein Crystallography, AcademicPress, New York.

Purified DAP or MDL can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to these antigens can be used as capture antibodies toimmobilize the respective DAP or MDL on the solid phase.

IX. Kits

This invention also contemplates use of DAP or MDL proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of DAP or MDL, or a bindingpartner. Typically the kit will have a compartment containing either adefined DAP or MDL peptide or gene segment or a reagent which recognizesone or the other.

A kit for determining the binding affinity of a test compound to, e.g.,a DAP12, would typically comprise a test compound; a labeled compound,for example a receptor complex binding partner or antibody having knownbinding affinity for the DAP12; a source of DAP12 (naturally occurringor recombinant); and a means for separating bound from free labeledcompound, such as a solid phase for immobilizing the DAP12. Oncecompounds are screened, those having suitable binding affinity to theDAP12 can be evaluated in suitable biological assays, as are well knownin the art, to determine whether they act as agonists or antagonists.The availability of recombinant DAP12 polypeptides also provide welldefined standards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., a DAP12, ina sample would typically comprise a labeled compound, e.g., antibody,having known binding affinity for the DAP12, a source of DAP12(naturally occurring or recombinant) and a means for separating thebound from free labeled compound, e.g., a solid phase for immobilizingthe DAP12. Compartments containing reagents, and instructions, willnormally be provided.

One method for determining the concentration of DAP12 in a sample wouldtypically comprise the steps of: (1) preparing membranes from a samplecomprised of a DAP12 source; (2) washing the membranes and suspendingthem in a buffer; (3) solubilizing the DAP12 by incubating the membranesin a culture medium to which a suitable detergent has been added; (4)adjusting the detergent concentration of the solubilized DAP12; (5)contacting and incubating said dilution with radiolabeled antibody toform complexes; (6) recovering the complexes such as by filtrationthrough polyethyleneimine treated filters; and (7) measuring theradioactivity of the recovered complexes.

Antibodies, including antigen binding fragments, specific for human DAPor DAP fragments are useful in diagnostic applications, e.g., to detectthe presence of elevated levels of DAP and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the DAP in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-partner complex) or heterogeneous (with aseparation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.For example, unlabeled antibodies can be employed by using a secondantibody which is labeled and which recognizes the antibody to a DAP orto a particular fragment thereof. These assays have also beenextensively discussed in the literature. See, e.g., Harlow and Lane(1988) Antibodies: A Laboratory Manual, CSH.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a human DAP, as such may be diagnostic of variousabnormal states. For example, overproduction of DAP may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody, or labeled DAP or MDL is provided.This is usually in conjunction with other additives, such as buffers,stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Any of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the test compound, DAP,MDL, or antibodies thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups: radiolabels suchas ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Both of the patents areincorporated herein by reference. Possibilities for indirect labelinginclude biotinylation of one constituent followed by binding to avidincoupled to one of the above label groups.

There are also numerous methods of separating the bound from the freebinding compound, or alternatively the bound from the free testcompound. The DAP or MDL can be immobilized on various matrices followedby washing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the DAP or MDL to a matrixinclude, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of antigen/binding compoundcomplex by any of several methods including those utilizing, e.g., anorganic solvent such as polyethylene glycol or a salt such as ammoniumsulfate. Other suitable separation techniques include, withoutlimitation, the fluorescein antibody magnetizable particle methoddescribed in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and thedouble antibody magnetic particle separation as described in U.S. Pat.No. 4,659,678.

The methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature. Many of thetechniques involve the use of activated carboxyl groups either throughthe use of carbodiimide or active esters to form peptide bonds, theformation of thioethers by reaction of a mercapto group with anactivated halogen such as chloroacetyl, or an activated olefin such asmaleimide, for linkage, or the like. Fusion proteins will also find usein these applications.

Another diagnostic aspect of this invention involves use ofpolynucleotide or oligonucleotide sequences taken from the sequence of aDAP or MDL. These sequences can be used as probes for detecting levelsof the antigen in samples from patients suspected of having an abnormalcondition, e.g., cancer or developmental problem. The preparation ofboth RNA and DNA nucleotide sequences, the labeling of the sequences,and the preferred size of the sequences has received ample descriptionand discussion in the literature. Normally an oligonucleotide probeshould have at least about 14 nucleotides, usually at least about 18nucleotides, and the polynucleotide probes may be up to severalkilobases. Various labels may be employed, most commonly radionuclides,particularly ³²P. However, other techniques may also be employed, suchas using biotin modified nucleotides for introduction into apolynucleotide. The biotin then serves as the site for binding to avidinor antibodies, which may be labeled with a wide variety of labels, suchas radionuclides, fluorescers, enzymes, or the like. Alternatively,antibodies may be employed which can recognize specific duplexes,including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, orDNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in any conventional techniques such as nucleicacid hybridization, plus and minus screening, recombinational probing,hybrid released translation (HRT), and hybrid arrested translation(HART). This also includes amplification techniques such as polymerasechain reaction (PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

X. Receptor Complex Partner

The description of the DAP and MDL proteins herein provide means toidentify receptor complex partners. Such receptor complex partner shouldbind specifically to the DAP12, DAP10, and/or MDL-1 with reasonably highaffinity. Various constructs are made available which allow eitherlabeling of the DAP or MDL to detect its partner. For example, directlylabeling DAP12, fusing onto it markers for secondary labeling, e.g.,FLAG or other epitope tags, Ig domain fusions, etc., will allowdetection of binding partners. This can be histological, as an affinitymethod for biochemical purification, or labeling or selection in anexpression cloning approach. A two-hybrid selection system may also beapplied making appropriate constructs with the available DAP12sequences. See, e.g., Fields and Song (1989) Nature 340:245-246.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

EXAMPLES I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York. Methods forprotein purification include such methods as ammonium sulfateprecipitation, column chromatography, electrophoresis, centrifugation,crystallization, and others. See, e.g., Ausubel, et al. (1987 andperiodic supplements); Deutscher (1990) “Guide to Protein Purification”in Methods in Enzymology, vol. 182, and other volumes in this series;and manufacturer's literature on use of protein purification products,e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif.Combination with recombinant techniques allow fusion to appropriatesegments, e.g., to a FLAG sequence or an equivalent which can be fusedvia a protease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QUIAGEN, Inc., Chatsworth, Calif.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; and Methods in Enzymology volumes. 70, 73, 74, 84, 92,93, 108, 116, 121, 132, 150, 162, and 163. Assays for neural cellbiological activities are described, e.g., in Wouterlood (ed. 1995)Neuroscience Protocols modules 10, Elsevier; Methods in NeurosciencesAcademic Press; and Neuromethods Humana Press, Totowa, N.J. Methodologyof developmental systems is described, e.g., in Meisami (ed.) Handbookof Human Growth and Developmental Biology CRC Press; and Chrispeels(ed.) Molecular Techniques and Approaches in Developmental BiologyInterscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

II. Amplification of Human DAP Fragment by PCR

Two primers are designed according to the provided sequences. Toincrease the chances of obtaining PCR products, human THP-1 cells, Th1 Tcells, monocytes activated with LPS, IFN-γ and IL-10, or NK cells areused. A product is purified, subcloned into pCR™ vector (Invitrogen, SanDiego Calif.), and then sequenced. See SEQ ID NO: 1, 2, 5, 6, 7, 8, 9,10, 11, 12, 13, and 14.

III. Tissue Distribution of Human DAP and MDL

Hybridization analysis or PCR analysis can be used. Preliminary data byhybridization suggests expression in macrophages, dendritic cells, someT cells, and NK cells. Analysis may be by Northern, Southern, or cDNANorthern techniques. Western blotting may be performed using appropriateantibodies or serum. Genomic sequences can also be determined bystandard techniques.

Southern blot analysis of human genomic DNA revealed a restrictionenzyme digest pattern consistent with the genomic organization of asingle DAP12 gene. Northern blot analysis indicated the abundantpresence of ˜0.7 kb DAP12 transcripts in peripheral blood leukocytes andspleen human, but not in thymus, prostate, testis, ovary, smallintestine or colon. DAP12 transcripts were detect in RNA isolated fromtwo human NK cell lines NKL and NK92, but not in the Jurkat T leukemiacell line or the JY EBV-transformed B lymphoblastoid cell line. Southernblot analysis of a large panel of cDNA libraries revealed predominantexpression of DAP12 in resting human peripheral blood mononuclear cells,dendritic cells (from which DAP12 was cloned), peripheral bloodmonocytes, and NK cell lines and clones.

Initial distribution data on DAP10 indicates that it is highly expressedin T cells, NK cells, monocytes, and dendritic cells. It does not appearto be highly expressed in EBV-transformed B cells.

The MDL-1 seems restricted in expression to monocytes, macrophages, anddendritic cells as analyzed by Southern blot analysis of a large panelof cDNA libraries and by RT-PCR. MDL-1 transcripts were not detected inT cells (pre-T cells, resting T cells, Th1 and Th2 T cell lines andclones), B cells, NK cells, granulocytes, mast cell lines, andendothelial cell lines. A panel of human fetal tissue librariesdisplayed hybridization with the fetal spleen library but with no otherlibrary, suggesting that the MDL-1 transcript is not expressed in celltypes of non-hematopoietic origin.

IV. Isolation of a Rodent DAP and MDL cDNA

SEQ ID NO: 5, 9, and 13 allow design of a probe or primer which willallow isolation of mouse counterparts. With the primate and rodentsequences, other species counterparts can be identified using conservedsequences, either nucleic acid or epitopes.

V. Sequencing of Isolated Clone

Standard methods are used to sequence a clone isolated as describedabove. The appropriate constructs for expression are prepare, e.g., in acoli, baculovirus, or mammalian cell type. Preferred cell types includeJurkat, YT, or Baf3. See ATCC catalog.

VI. Expression of Human DAP and MDL Protein

Soluble DAP12-FLAG protein is transiently expressed in COS-7 cells. Arecombinant form of DAP12 displaying the FLAG peptide at the amino orcarboxy terminus (Hoppe, et al. (1988) Biotechnology 6:1205-1210) isintroduced into the expression vector pME18S and subsequentlytransfected into COS-7 cells by electroporation. Electroporated cellsare grown in DMEM medium supplemented either with 1% Nutridoma HU(Boehringer Mannheim, Mannheim, Germany) or DMEM medium alone. Similarmethods are used for the DAP10 or MDL-1.

VII. Purification of Soluble DAP FLAG Protein

Supernatant containing soluble DAP12 FLAG is passed on a 20 ml column ofCu⁺⁺ ions attached to a Chelating Sepharose Fast Flow matrix (Pharmacia,Upsalla, Sweden). After washing with binding buffer (His-Bind Bufferkit, Novagen, Madison, Wis.), the proteins retained by the metal ionsare eluted with a gradient of Imidazole. The content of human DAP12 FLAGin the eluted fractions is determined, e.g., by dot blot using theanti-FLAG monoclonal antibody M2 (Eastman Kodak, New Haven, Conn.) or byCoomassie blue and silver staining of reducing SDS-PAGE. The DAP12 FLAGprotein containing fractions is then pooled and dialyzed against PBS.

VIII. Stable Expression of Membrane DAP or MDL

A native membrane form is subcloned into an expression vector, e.g.,pMAMneo (Clontech, Palo Alto, Calif.), which contains the RSV-LTRenhancer linked to the dexamethasone-inducible MMTV-LTR promoter. Thisconstruct is then transfected into NIH-3T3 cells by electroporation.Transfected NIH-3T3 cells are seeded in selective 0.5 mg/ml Geneticin(G418; Boehringer-Mannheim, Mannheim, Germany) DMEM supplemented with10% Fetal Calf Serum.

Biochemical characterization of membrane DAP12 protein in stabletransfected NIH-3T3 cells may be performed with metabolic labeling.Cells are cultivated, e.g., in DMEM medium supplemented with 10% FetalCalf Serum and 1 μM final dexamethasone (Sigma, Saint Quentin Fallavier,France). Cells are then incubated with ³⁵S-Met and ³⁵S-Cys to labelcellular proteins. Analysis of the proteins under reducing conditions onSDS-PAGE should show a 12 kDa protein, but not in the lysate ofuntransfected NIH-3T3 cells. Certain other structural features areknown, e.g., glycosylation sites, etc.

IX. Preparation of Antibodies Specific for DAP

Inbred Balb/c mice are immunized intraperitoneally with recombinantforms of the primate protein. Animals are boosted at appropriate timepoints with protein, with or without additional adjuvant, to furtherstimulate antibody production. Serum is collected, or hybridomasproduced with harvested spleens.

Alternatively, Balb/c mice are immunized with cells transformed with thegene or fragments thereof, either endogenous or exogenous cells, or withisolated membranes enriched for expression of the antigen. Serum iscollected at the appropriate time, typically after numerous furtheradministrations. Various gene therapy techniques may be useful, e.g., inproducing protein in situ, for generating an immune response.

Monoclonal antibodies may be made. For example, splenocytes are fusedwith an appropriate fusion partner and hybridomas are selected in growthmedium by standard procedures. Hybridoma supernatants are screened forthe presence of antibodies which bind to the human DAP12, e.g., by ELISAor other assay. Antibodies which specifically recognize human DAP12 butnot species variants may also be selected or prepared.

In another method, synthetic peptides or purified protein are presentedto an immune system to generate monoclonal or polyclonal antibodies.See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. In appropriate situations, the binding reagent is eitherlabeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. Nucleic acids may alsobe introduced into cells in an animal to produce the antigen, whichserves to elicit an immune response. See, e.g., Wang, et al. (1993)Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

Antibodies have been made, and used, as described below, for both theDAP proteins.

X. Mapping of Human DAP

Chromosome spreads are prepared. In situ hybridization is performed onchromosome preparations obtained from phytohemagglutinin-stimulatedhuman lymphocytes cultured for 72 h. 5-bromodeoxyuridine was added forthe final seven hours of culture (60 μg/ml of medium), to ensure aposthybridization chromosomal banding of good quality.

A PCR fragment, amplified with the help of primers, is cloned into anappropriate vector. The vector is labeled by nick-translation with ³H.The radiolabeled probe is hybridized to metaphase spreads at finalconcentration of 200 ng/ml of hybridization solution as described inMattei, et al. (1985) Hum. Genet. 69:327-331.

After coating with nuclear track emulsion (KODAK NTB₂), slides areexposed. To avoid any slipping of silver grains during the bandingprocedure, chromosome spreads are first stained with buffered Giemsasolution and metaphase photographed. R-banding is then performed by thefluorochrome-photolysis-Giemsa (FPG) method and metaphasesre-photographed before analysis.

The genomic organization of human DAP12 consists of 5 exons spanning ˜4kb on chromosome 19q13.1. The human KIR genes (Baker, et al. (1995)Chromosome Research 3:511) and the related LAIR (Meyaard, et al. (1997)Immunity 7:283-290, and ILT/MIR (Wagtmann, et al. (1997) Current Biology7:615-618) genes are all located nearby on chromosome 19q13.4.

XI. DAP and MDL Biology

DAP12 is a disulfide-bonded homodimer, containing an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain, thatis predominantly expressed in NK cells, monocytes, and dendritic cells.This molecule non-covalently associates with membrane glycoproteins ofthe killer cell inhibitory receptor (KIR) family that lackimmunoreceptor tyrosine-based inhibitory motifs (ITIM) in theircytoplasmic domain. Cross-linking KIR2DS2-DAP12 complexes expressed intransfectants results in cellular activation, as demonstrated bytyrosine-phosphorylation of cellular proteins and up-regulation of earlyactivation antigens. Phosphorylated DAP12 peptides bind ZAP-70 and Sykprotein tyrosine kinases, suggesting an activation pathway similar tothe T and B cell antigen receptors.

NK cells express membrane receptors of the immunoglobulin and C-typelectin superfamilies that recognize MHC class I and inhibit NKcell-mediated cytotoxicity. Lanier (1997) Immunity 6:371-378. Theseinhibitory receptors (including human KIR, human CD94/NKG2A, and rodentLy49) possess ITIM in their cytoplasmic domains that recruit SH2-domaincontaining protein tyrosine phosphatases (SHP) 1 or 2, resulting ininactivation of NK cell function. Burshtyn, et al. (1996) Immunity4:77-85; Olcese, et al. (1996) J. Immunol. 156:4531-4534; and Houchins,et al. (1997) J. Immunol. 158:3603-3609. Certain isoforms of the KIR,Ly49, and CD94/NKG2 receptors lack ITIM sequences and it has beenproposed that these ‘non-inhibitory’ receptors may activate, rather thaninhibit, NK cell function. Houchins, et al. (1997) J. Immunol.158:3603-3609; Biassoni, et al. (1996) J. Exp. Med. 183:645-650; andMason, et al. (1996) J. Exp. Med. 184:2119-2128. When the non-inhibitoryKIR2DS2 molecule was expressed by transfection in the RBL-2H3 basophilicleukemia no cellular activation was observed when the receptors wereligated, suggesting that these ‘non-inhibitory’ NK receptors may lackintrinsic signaling properties. Bléry, et al. (1997) J. Biol. Chem.272:8989-8996.

Recently, Olcese, et al. (1997) J. Immunol. 158:5083-5086, reported thatan unknown phosphoprotein of ˜12 kD, expressed as a disulfide-bondeddimer, was co-immunoprecipitated with a non-inhibitory KIR2DS2glycoprotein from NK cell lysates. Cell surface Ig receptors, T cellantigen receptors (TcR), and certain Fc receptors (FcR) non-covalentlyassociate with small transmembrane proteins (e.g. CD3δ, γ, ε, ζsubunits, CD79α, β, FcεRI-γ) containing ITAM sequences(D/ExxYxxL/I-x₆₋₈-YxxL/I; Reth (1989) Nature 338:383-384) that arerequired for signal transduction by these receptor complexes. Chan, etal. (1994) Ann. Rev. Immunol. 12:555-592. Therefore, it seems likelythat these non-inhibitory NK cell receptors might require an associatedprotein with similar properties to mediate positive signal transduction.

A database of expressed tag sequences (EST) from a large panel of cDNAlibraries was searched with a TBLASTN algorithm program for moleculesbearing homology with the human CD3δ, γ, ε, ζ and FcεRI-γ proteinsequences. An EST from a human CD1+ dendritic cell library was selectedfor further study based on identification of an ITAM in this molecule.Sequencing of the 604 bp cDNA revealed an open reading frame of 339nucleotides, encoding a putative type I membrane protein of 113 aminoacids (see SEQ ID NO: 1 and 2). The protein, designated DAP12, iscomposed of a 27 aa leader, 14 aa extracellular domain, 24 aatransmembrane segment, and 48 aa cytoplasmic region. Although DAP12 hasless than 25% homology with the human CD3δ, γ, ε, ζ and FcεRI-γproteins, the cytoplasmic domain contains the peptide,ESPYQELQGQRSDVYSDL (see SEQ ID NO: 2), that precisely corresponds to theprototype ITAM consensus sequence. Potential sites for phosphorylationby protein kinase C (residues 79-81 and 107-109) and casein kinase II(residues 85-88) are also present in the DAP12 cytoplasmic region. Thetransmembrane region contains a charged amino acid (D), also conservedin the transmembrane domain of the CD3 subunits. A potential murinehomolog of DAP12 is ˜70% homologous with the human DAP12 protein and hasa conserved D residue in the transmembrane region, conserved C residuesin the extracellular domain and an ITAM in the cytoplasmic region.

A conspicuous feature of the non-inhibitory KIR (Biassoni, et al. (1996)J. Exp. Med. 183:645-650), Ly49D and Ly49H (Mason, et al. (1996) J. Exp.Med. 184:2119-2128), CD94 (Chang, et al. (1995) Eur. J. Immunol.25:2433-2437), NKG2C and NKG2E (Houchins, et al. (1991) J. Exp. Med.173:1017-1020), and ILT1 (Samaridis and Colonna (1997) Eur. J. Immunol.27:660-665) receptors is the presence of a basic amino acid (K or R) inthe transmembrane domain. Given the precedent for interactions betweenproteins of multi-subunit receptor complexes via oppositely chargedamino acids in the transmembrane domains, e.g. the CD3/TcR complex(Chan, et al. (1994) Ann. Rev. Immunol. 12:555-592), we examined whetherDAP12 associates with the non-inhibitory KIR2DS2 glycoprotein containinga K in the transmembrane region (Colonna and Samaridis (1995) Science268:405-408). The murine Ba/F3 pre-B cell line was transfected with acDNA encoding KIR2DS2 either alone or together with a DAP12 cDNAcontaining a FLAG epitope tag at the N terminus to permit detection withan anti-FLAG mAb. Transfectants were selected by flow cytometry for cellsurface expression based on positive staining with anti-KIR mAb DX27 oranti-FLAG mAb M2. KIR2DS2 Ba/F3 and KIR2DS2+DAP12-FLAG Ba/F3transfectants were surface labeled with ¹²⁵I, lysed in 1% digitonin topreserve non-covalent associations of membrane protein complexes, andimmunoprecipitated with anti-KIR mAb or anti-FLAG mAb. The tyrosineresidue in the FLAG epitope provided a site for radioiodination,permitting visualization of the DAP12 protein. Anti-KIR mAbimmunoprecipitated an ¹²⁵I labeled species of ˜50-60 kD from both theKIR2DS2 Ba/F3 cells and KIR2DS2+DAP12-FLAG Ba/F3 transfectants,consistent with the predicted molecular weight of the KIR2DS2glycoprotein. An additional ¹²⁵I labeled protein of ˜12 kD wasco-immunoprecipitated with anti-KIR mAb from the KIR2DS2+DAP12-FLAGtransfectant, but not from the transfectant expressing only KIR2DS2.Reciprocally, an ¹²⁵I labeled glycoprotein migrating identical toKIR2DS2 was co-immunoprecipitated with anti-FLAG mAb from theKIR2DS2+DAP12-FLAG Ba/F3 cells, but not from the KIR2DS2 onlytransfectant. Comparison of immunoprecipitates analyzed by SDS-PAGEusing either reducing or non-reducing conditions indicate that DAP12 isexpressed on the cell surface as a disulfide-bonded dimer. It should benoted that we were unable to detect cell surface expression of DAP12 onthe surface of Ba/F3 cells transfected with the DAP12-FLAG cDNA alone,without KIR2DS2. However, DAP12-FLAG proteins were detected in thecytoplasm, suggesting that DAP12 may require association with itspartner subunits for efficient transport to the cell surface, similar tothe situation with the CD3 proteins (Clevers, et al. (1988) Ann. Rev.Immunol. 6:629-662). Additionally, preliminary results indicated thatDAP12 does not associate with the inhibitory KIR isoforms that lack acharged residue in their transmembrane domain.

A peptide corresponding to the cytoplasmic domain of DAP12(ITETESPY*QELQGQRSDVY*SDLNTQRP; see SEQ ID NO: 2) was synthesized eitheras an unphosphorylated protein or containing phosphates on both Yresidues. Lysates from Jurkat T cells or NK cell clone A6 were incubatedwith the biotinylated peptides and complexes precipitated usingavidin-agarose. Western blot analysis demonstrated that a DAP12 peptidephosphorylated on both Y residues, but not the unphosphorylated peptide,formed complexes with the ZAP-70 kinase. The tyrosine phosphorylatedDAP12 peptide, but not the unphosphorylated DAP12 peptide, also formed acomplex with the Syk protein tyrosine kinase in lysates from NK cells.The binding of these kinases to phosphorylated DAP12 is remarkablyreminiscent of the interactions that have been demonstrated between thephosphorylated ITAM-containing CD3 subunits and Syk or ZAP-70 kinasesduring TcR signaling. Iwashima, et al. (1994) Science 263:1136-1139; andChan, et al. (1994) J. Immunol. 152:4758-4766.

Ligation of the CD3/TcR complex on T cells or the Ig receptor complex onB cells resulted in cellular activation. Therefore, studies wereundertaken to examine the functional consequence of cross-linking theKIR2DS2-DAP12 complex. Ba/F3 transfectants expressing either KIR2DS2alone or the KIR2DS2-DAP12-FLAG complex were incubated with anti-KIR mAbDX27 or anti-FLAG mAb, followed by a goat anti-mouse Ig to providecross-linking. Examination of total cellular proteins in Ba/F3 cellsexpressing the KIR2DS2-DAP12-FLAG complex that were stimulated withanti-KIR or anti-FLAG mAb revealed tyrosine phosphorylation of severalcellular substrates. Immunoprecipitation with anti-FLAG mAb and Westernblot analysis with anti-phosphotyrosine mAb demonstrated thatcross-linking the KIR2DS2-DAP12-FLAG transfectants with anti-KIR mAbinduced tyrosine phosphorylation of the DAP12 protein and resulted inthe association of phosphorylated DAP12 with the Syk protein tyrosinekinase. By contrast, Ba/F3 cells expressing only KIR2DS2 were notactivated by cross-linking with anti-KIR mAb. Similarly, up-regulationof CD69 expression was observed in Jurkat T leukemia cells transfectedwith both KIR2DS2 and DAP12, but not KIR2DS2 alone, when these receptorswere cross-linked with anti-KIR mAb. These results indicate that DAP12is necessary and responsible for KIR2DS2 signal transduction in thesehost cells and are in accordance with prior observations demonstratingthat KIR2DS2 molecules are functional in NK cells, but not intransfectants expressing only KIR2DS2. Bléry, et al. (1997) J. Biol.Chem. 272:8989-8996.

These studies suggest that DAP12 may associate with the non-inhibitoryisoforms of the KIR molecules in NK cells and permit cellular activationvia these receptors, similar to the function of the CD3 subunits in theTcR complex and CD79 subunits in the B cell receptor complex. Expressionof DAP12 in monocytes and dendritic cells predicts association withother receptors similar to the non-inhibitory KIR present in these celltypes. Likely candidates are the recently identified ILT4MIR family ofmolecules expressed by human monocytes (Wagtmann, et al. (1997) CurrentBiology 7:615-618; and Samaridis and Colonna (1997) Eur. J. Immunol.27:660-665) and the PIR-A molecules in rodent myeloid and B cells(Hayami, et al. (1997) J. Biol. Chem. 272:7320-7327; and Kubagawa, etal. (1997) Proc. Natl. Acad. Sci. USA 94:5261-5266). In addition, thephysical properties of DAP12 are similar to a novel dimeric 12 kDphosphoprotein identified in the pre-T cell receptor complex on murinethymocytes. Takase, et al. (1997) J. Immunol. 159:741-747. Thus, DAP12may function in cellular activation mediated by a diverse array ofreceptors in distinct cell lineages.

Cloning and Sequence Analysis

TBLASTN searches of the DNAX sequence database were made using the humanCD3δ, γ, ε, ζ and FcεRI-γ protein sequences. The cDNA insert in plasmidLL603, identified in a human CD1+ dendritic cell library, was isolatedand subjected to automated sequencing (ABI).

DNA and RNA

RNA from human tissues and human genomic DNA were purchased fromClontech (Palo Alto, Calif.). Northern and Southern blot analysis wereperformed as described. Chang, et al. (1995) Eur. J. Immunol.25:2433-2437.

Transfection

A cDNA containing the CD8 leader segment, followed by the FLAG peptideepitope, and joined to the extracellular, transmembrane and cytoplasmicsegments of DAP12 was subcloned into the pMX-puro retroviral vector(Onihsi, et al. (1996) Exp. Hematology 24:324-329; generously providedby Dr. T. Kitamura, DNAX), packaged using the Phoenix cell line (kindlyprovided by Dr. G. Nolan, Stanford), and virus was used to infect themouse pre-B cell line Ba/F3 (Onihsi, et al. (1996) Exp. Hematology24:324-329). The NKAT5 cDNA (Colonna and Samaridis (1995) Science268:405-408) encoding KIR2DS2 (kindly provided by Dr. M. Colonna, Basel)was subcloned into the pMX-neo retroviral vector. Ba/F3 cells wereinfected, drug selected, and transfectants isolated using flowcytometry. Onihsi, et al. (1996) Exp. Hematology 24:324-329. DAP12 cDNAwas subcloned into the pEF-BOS vector for transient expression in Jurkatcells using electroporation for introduction of the plasmid. Wu, et al.(1995) Mol. Cell. Biol. 15:4337-4346.

Immunoprecipitation.

Cells were labeled with ¹²⁵I and solubilized in lysis buffer (pH 7.8, 1%digitonin (Sigma), 0.12% Triton-X100, 150 mM NaCl, 20 mMtriethanolamine, 0.01% NaN₃, and protease inhibitors). Lanier, et al.(1989) Nature 342:803-805. Cell lysates were incubated on ice for 2 hrwith Pansorbin (Calbiochem) coated with rabbit anti-mouse Ig (Sigma) andmouse anti-KIR2D mAb DX27, anti-FLAG mAb M2 (Kodak), or control IgG andthen washed in Tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl, pH8.0) containing 5 mM CHAPS (Sigma) and protease inhibitors. Lanier, etal. (1989) Nature 342:803-805. Biotinylated peptides corresponding toresidues ITETESPY*QELQGQRSDVY*SDLNTQRP in the cytoplasmic domain ofDAP12 (see SEQ ID NO: 2) were synthesized, either unphosphorylated orcontaining phosphate on both Y residues (generously provided by Dr. C.Turck, UCSF). Control unphosphorylated and Y-phosphorylated CD3ζpeptides (Iwashima, et al. (1994) Science 263:1136-1139) were a giftfrom Dr. A. Weiss (UCSF). Biotinylated peptides were incubated withlysates from Jurkat or NK clone A6 cells, precipitated withavidin-agarose, and washed in Tris-buffered saline (50 mM Tris, 150 mMNaCl, pH 7.8) containing 1% NP-40 and protease inhibitors (Iwashima, etal. (1994) Science 263:1136-1139). Immunoprecipitates were analyzed byWestern blot (Phillips, et al. (1996) Immunity 5:163-172) usinganti-ZAP-70 mAb or rabbit anti-Syk specific antiserum (Iwashima, et al.(1994) Science 263:1136-1139; kindly provided by Art Weiss, UCSF).

Cell Activation

Ba/F3 cells expressing either KIR2DS2 alone, DAP12 (FLAG epitope tagged)alone, or the KIR2DS2-DAP12 complex were incubated with the indicatedmAbs at 4° C., washed, and then cross-linked with F(ab′)2 goatanti-mouse Ig for 3 min at 37° C. Cells were lysed in TBS containing 1%NP-40 and protease inhibitors. Total cell lysates or immunoprecipitatesof DAP12-FLAG with anti-FLAG mAb M2 were analyzed by Western blot usingHRP-conjugated anti-phosphotyrosine mAb 4G10 (UBI). Jurkat cells stablytransfected with the NKAT5 cDNA (Colonna and Samaridis (1995) Science268:405-408) using a retroviral vector (Onihsi, et al. (1996) Exp.Hematology 24:324-329) were transiently transfected by electroporationwith human DAP12 cDNA in the pEF-BOS vector or sham-transfected with acontrol vector. Wu, et al. (1995) Mol. Cell. Biol. 15:4337-4346. After24 hours, transfectants were incubated in microtiter plates pre-coated(5 μg/ml) with control Ig or anti-KIR mAb DX27. After 12 hr incubation,transfectants were harvested and then stained with FITC conjugatedanti-CD69 or control mAb and analyzed by flow cytometry. Lanier andRecktenwald (1991) Methods: A Companion to Methods in Enzymology2:192-199.

XII. DAP12 Associates with Activating CD94/NKG2C NK Cell Receptors

While the inhibitory NK cell receptors for MHC class I expressImmunoreceptor Tyrosine-based Inhibitory Motifs (ITIM) that recruitintracellular tyrosine phosphatases and prevent NK cell effectorfunction, the activating NK cell receptors lack intrinsic sequencesrequired for cellular stimulation. CD94/NKG2C, an activating NK cellreceptor of the C-type lectin superfamily which binds to HLA-E,non-covalently associates with DAP12, a membrane receptor containing anImmunoreceptor Tyrosine-based Activating Motif (ITAM). Efficientexpression of CD94/NKG2C on the cell surface requires the presence ofDAP12 and charged residues in the transmembrane domains of DAP12 andNKG2C are necessary for this interaction. These results provide amolecular basis for the assembly of NK cell receptors for MHC class Iinvolved in cellular activation and inhibition.

NK cells are lymphocytes that participate in innate immune responsesagainst certain bacteria, parasites, and viruses (reviewed in Scott andTrinchieri (1995) Current Opinion Immunol. 7:34-40; Trinchieri (1989)Adv. Immunol. 47:187-376). How NK cells recognize pathogens is unclear;however, one aspect of this process may involve the detection andelimination of host cells that have lost or down-regulated expression ofMHC class I as a consequence of infection. NK cells express receptorsfor MHC class I that can either activate or inhibit cell-mediatedcytotoxicity and cytokine production (reviewed in Lanier, (1998) Cell92:705-707; Lanier (1998) Ann. Rev. Immunol. 16:359-393). Several typesof NK cell receptors for MHC class I have been identified (Lanier (1998)Cell 92:705-707). In humans, the Killer Cell Inhibitory Receptors (KIR)comprise a small family of molecules encoded by genes of the Igsuperfamily (Colonna and Samaridis (1995) Science 268:405-408; D'Andrea,et al. (1995) J. Immunol. 155:2306-2310; Wagtmann, et al. (1995)Immunity 2:439-449). Within the KIR family, certain isoforms possess twoIg-domains (KIR2D) or three Ig-domains (KIR3D) in the extracellularregion that are involved in recognition of polymorphic HLA-C or HLA-Bligands, respectively (Dohring and Colonna (1996) Eur. J. Immunol.26:365-369; Fan, et al. (1996) Proc. Nat'l Acad. Sci. USA 93:7178-7183;Litwin, et al. (1994) J. Exp. Med. 180:537-543; Rajagopalan and Long(1997) J. Exp. Med. 185:1523-1528; Rojo, et al. (1997) Eur. J. Immunol.27:568-571; and Wagtmann, et al. (1995) Immunity 3:801-809).Heterogeneity also exists in the transmembrane and cytoplasmic domainsof different KIR molecules. Upon ligand binding, KIR having ITIM intheir cytoplasmic domain (designated KIR2DL and KIR3DL) recruit SHP-1and prevent NK cell effector function (Burshtyn, et al. (1996) Immunity4:77-85; Campbell, et al. (1996) J. Exp. Med. 184:93-100; Fry, et al.(1996) J. Exp. Med. 184:295-300; and Olcese, et al. (1996) J. Immunol.156:4531-4534). In contrast, KIR isoforms lacking ITIM and having abasic K amino acid in the transmembrane (KIR2DS and KIR3DS) have beenimplicated in NK cell activation (Biassoni, et al. (1996) J. Exp. Med.183:645-650; Olcese et al. (1997) J. Immunol. 158:5083-5086). KIR2DS arenon-covalently associated with an ITAM-bearing adapter molecule, DAP12,that is expressed on the surface of NK cells as a disulfide-bondedhomodimer (Campbell, et al. (1998) Eur. J. Immunol. 28:599-609; Lanier(1998) Cell 92:705-707; Olcese, et al. (1997) J. Immunol.158:5083-5086). Upon cross-linking of KIR2DS, tyrosine residues in theITAM of DAP12 become phosphorylated and recruit ZAP-70 or Syk, resultingin cellular activation (Lanier (1998) Cell 92:705-707). Human DAP12 ispresent on human chromosome 19q13.1 near the KIR gene family (Baker, etal. (1995) Chromosome Research 3:511), demonstrating a genetic linkagebetween KIR and DAP12.

Another type of NK cell receptor, CD94/NKG2, is a heterodimer composedof an invariant CD94 glycoprotein that is disulfide-bonded to either aNKG2A or a NKG2C glycoprotein (Brooks, et al. (1997) J. Exp. Med.185:795-800; Carretero, et al. (1997) Eur. J. Immunol. 27:563-575;Lazetic, et al. (1996) J Immunol. 157:4741-4745). CD94 (Chang, et al.(1995) Eur. J. Immunol. 25:2433-2437) and four NKG2 genes (NKG2A, NKG2C,NKG2E, and NKG2D/F; Houchins, et al. (1991) J. Immunol. 158:3603-3609;and Plougastel and Trowsdale (1997) Eur. J. Immunol. 27:2835-2839) areall members of the C-type lectin superfamily and are closely linked onhuman chromosome 12p12-p13 in the “NK complex” (Renedo, et al. (1997)Immunogenetics 46:307-311). Rodent homologs of the human CD94 and NKG2genes are located in the “NK complex” on mouse and rat chromosomessyntenic with human chromosome 12 (Berg, et al. (1998) Eur. J. Immunol.28:444-450; Dissen, et al. (1997) Eur. J. Immunol. 27:2080-2086; andVance, et al. (1997) Eur. J. Immunol. 27:3236-3241).

Antibodies against CD94 can either activate or inhibit NK cell-mediatedcytotoxicity against Fc-receptor bearing targets and different NK cellclones isolated from a single individual demonstrate heterogeneousbehavior in these functional assays (Brumbaugh, et al. (1996) J.Immunol. 157:2804-2812; Perez-Villar, et al. (1996) J. Immunol.157:5367-5374; and Pérez-Villar et al. (1995) J. Immunol.154:5779-5788). This phenomenon was explained by the finding that CD94forms disulfide-linked heterodimers with either NKG2A or NKG2C (Brooks,et al. (1997) J. Exp. Med. 185:795-800; Cantoni, et al. (1998) Eur. J.Immunol. 28:327-338; Carretero, et al. (1997); and Lazetic, et al.(1996) J Immunol. 157:4741-4745). NKG2A contains an ITIM sequence in thecytoplasmic domain that upon receptor ligation becomes tyrosinephosphorylated and recruits SHP-1 or SHP-2 which in turn inhibit NKeffector function (Houchins, et al. (1997) J. Immunol. 158:3603-3609;and Le Drean, et al. (1998) Eur. J. Immunol. 28:264-276). In contrast,NKG2C lacks an ITIM and receptor ligation results in NK cell activation(Cantoni, et al. (1998) Eur. J. Immunol. 28:327-338; and Houchins, etal. (1997) J. Immunol. 158:3603-3609). CD94 is necessary to transportboth NKG2A and NKG2C to the cell surface (Lazetic, et al. (1996) JImmunol. 157:4741-4745). Within the NK cell population in an individual,CD94/NKG2A and CD94/NKG2C receptors are expressed on overlappingsubpopulations and some NK cells may express CD94 proteins that are notassociated with either NKG2A or NKG2C (Cantoni, et al. (1998) Eur. J.Immunol. 28:327-338). Thus, CD94 and the NKG2 proteins can form adiverse receptor repertoire in an individual. CD94/NKG2A and CD94/NKG2Creceptors recognize HLA-E (Borrego, et al. (1998) J. Exp. Med.187:813-818; Braud, et al. (1998) J. Immunol. 159:5192-5196), anon-classical MHC class I molecule that has the unique property ofbinding 9 amino acid peptides derived from the leader segments of otherclassical HLA class I proteins (Braud, et al. (1997) Eur. J. Immunol.27:1164-1169). While the ITIM in NKG2A explains the inhibitory functionof the CD94/NKG2A receptor, neither CD94 nor NKG2C possess sequences intheir cytoplasmic domains that provide for intrinsic signaling capacity.However, the existence of a basic amino acid in the transmembrane ofNKG2C suggested possible interactions with the DAP12 receptor.

Association of DAP12 with CD94/NKG2C Receptors

To determine whether DAP12 might be associated with the activatingCD94/NKG2C receptor complex, A mouse pre-B cell line, Ba/F3, wasco-infected with ecotropic retroviruses encoding human CD94, NKG2C, andDAP12 (containing a FLAG epitope on the N-terminus to permit detectionon the cell surface). Consistent with prior results (Lanier (1998) Cell92:705-707), transfection of FLAG-DAP12 alone into Ba/F3 cells does notpermit cell surface expression of this receptor, although FLAG-DAP12proteins were detected in the cytoplasm of these transfectants asdetermined by cytoplasmic staining and Western blot analysis. Similarly,cell surface expression of NKG2C alone in Ba/F3 cells or inFLAG-DAP12+Ba/F3 transfectants co-infected with NKG2C could was notdetected. In contrast, CD94 alone was expressed on the cell surface ofBa/F3 cells. However, CD94 is not competent to transport FLAG-DAP12 tothe cell surface in Ba/F3 cells co-infected with both CD94 andFLAG-DAP12, although FLAG-DAP12 was detected in the cytoplasm of thesetransfectants by Western blot and cytoplasmic immunofluorescence.Furthermore, when CD94+ Ba/F3 cells were infected with a retrovirusencoding NKG2C, CD94/NKG2C heterodimers on the cell surface, using anantiserum that detects the CD94/NKG2C complex were not detected (Braud,et al. (1998) J. Immunol. 159:5192-5196; Lazetic, et al. (1996) J.Immunol. 157:4741-4745) (although it is possible to obtain low levels ofsurface expression of CD94/NKG2C heterodimers using episomal vectorscontaining strong promoters in highly efficient transfection systemssuch as 293T cells; Braud, et al. (1998) J. Immunol. 159:5192-5196;Lazetic, et al. (1996) J Immunol. 157:4741-4745). When Ba/F3 cells wereinfected with retroviruses encoding human CD94, NKG2C, and FLAG-DAP12,expression of FLAG-DAP12 and a CD94/NKG2C receptor on the cell surfaceof the CD94/NKG2C/DAP12 transfectants were detected. Collectively, theseexperiments support the existence of a multi-subunit receptor complexcomposed of CD94, NKG2C, and DAP12.

Ba/F3 transfectants expressing CD94, NKG2C, and FLAG-DAP12 were labeledwith ¹²⁵I, solubilized in digitonin detergent to preserve non-covalentmembrane receptor complexes (Lanier, et al. (1989) Nature 342:803-805),and immunoprecipitated with antibodies against human CD94 or FLAG.Immunoprecipitation with anti-CD94 from the CD94/NKG2C/FLAG-DAP12 Ba/F3transfectants revealed ¹²⁵I labeled proteins consistent with thepredicted mobility of NKG2C and FLAG-DAP12. It has been previouslyreported that human CD94 does not label efficiently with ¹²⁵I (Lazetic,et al. (1996) J Immunol. 157:4741-4745; Phillips, et al. (1996) Immunity5:163-172), so the 40 kD radiolabeled subunit immunoprecipitated withanti-CD94 mAb represents a NKG2C glycoprotein that is disulfide-bondedto CD94 (Lazetic, et al. (1996) J Immunol. 157:4741-4745). When analyzedusing non-reducing conditions, FLAG-DAP12 migrated predominately as adisulfide-bonded homodimer and the mobility of NKG2C was consistent withthe existence of a CD94/NKG2C heterodimer. Therefore, it appears thatthe minimal CD94/NKG2C-DAP12 receptor complex may be a tetramercomprised of a disulfide-linked DAP12 homodimer non-covalentlyassociated with a disulfide-linked CD94/NKG2C heterodimer.

XIII. DAP12 is Required for Cell Surface Expression of CD94/NKG2C UsingCharged Residues in the Transmembrane Domains of DAP12 and NKG2C

The Role of Charged Amino Acids in the Transmembrane of KIR, NKG2, andDAP12 Receptors in the Assembly of the Multi-Subunit Complexes

The NKG2A and NKG2C proteins demonstrate 75% amino acid identity(Houchins, et al. (1991) J. Immunol. 158:3603-3609) and both CD94/NKG2Aand CD94/NKG2C receptors bind to a common ligand, HLA-E (Braud, et al.(1998) J. Immunol. 159:5192-5196). A conspicuous difference betweenNKG2A and NKG2C is the presence of a basic residue in the transmembraneof NKG2C that is absent in NKG2A and CD94. In contrast to NKG2C,infection of CD94+ Ba/F3 cells with a retrovirus encoding human NKG2Apermits expression of a CD94/NKG2A complex on the cell surface in theabsence of DAP12. The presence of a CD94/NKG2A complex on Ba/F3 cellsdoes not permit expression of FLAG-DAP12 on the cell surface, althoughFLAG-DAP12 proteins were detected in the cytoplasm of thesetransfectants by immunofluorescence and Western blot analysis.

Because other multi-subunit membrane receptors have been shown toassociate via salt bridges formed by acidic and basic amino acids intheir transmembranes (e.g., CD3/TcR (Bonifacino, et al. (1991) EMBO J.10:2783-2793; Cosson, et al. (1991) Nature 351:414-416; Morley, et al.(1988) J. Exp. Med. 168:1971-1978), the requirement of the D residue inDAP12 was examined for association with CD94/NKG2C. The D residue inFLAG-DAP12 was converted to A by site-directed mutagenesis and thismutant receptor was transfected into Ba/F3 cells. Unlike wild-typeFLAG-DAP12, the D-A transmembrane FLAG-DAP12 mutant receptor wasexpressed on the cell surface in the absence of other subunits,indicating that the D residue in the transmembrane serves as a retentionsignal for DAP12, similar to the function of the charged residues in thetransmembrane of the CD3 proteins (Bonifacino, et al. (1990) Cell63:503-513; Bonifacino, et al. (1991) EMBO J. 10:2783-2793; Cosson, etal. (1991) Nature 351:414-416). As noted previously, Ba/F3 cellstransfected with CD94 and NKG2C do not efficiently express a CD94/NKG2Cheterodimer on the cell surface in the absence of DAP12. Infection ofthese CD94/NKG2C+Ba/F3 transfectants with the D-A transmembraneFLAG-DAP12 mutant receptor did not permit efficient expression ofCD94/NKG2C on the cell surface, as indicated by the marginal reactivityof these cells with an anti-CD94/NKG2 specific antisera (although NKG2Cproteins were detected in the cytoplasm of the transfectant by Westernblot analysis).

Comparison of the transmembrane domains of NKG2A and NKG2C indicates thepresence of a K residue in NKG2C, suggesting this residue may beresponsible for interaction with the D residue in DAP12. Therefore, theK in NKG2C was converted to L by site-directed mutagenesis and the K-Ltransmembrane NKG2C mutant was transfected into Ba/F3 cells expressingDAP12 and CD94. Ba/F3 cells co-transfected with CD94 and the K-Ltransmembrane NKG2C mutant expressed did not permit surface expressionof FLAG-DAP12, although DAP12 was detected in the cytoplasm by Westernblot analysis. Very low levels of a CD94/K-L transmembrane NKG2C mutantreceptor were detected on the surface of these transfectants using ananti-CD94/NKG2C antiserum. Although the K residue in the transmembraneof NKG2C might serve as a retention signal, it should be noted thatNKG2C also expresses the motif DxxxLL that is also present in CD3γ andhas been implicated in the degradation, transport and localization ofCD3 proteins (Dietrich, et al. (1994) EMBO J. 13:2156-2166; Dietrich, etal. (1997) J. Cell Biol. 138:271-281; Dietrich, et al. (1996) J. CellBiol. 132:299-310; Letourneur and Klausner (1992) Cell 69:1143-1157) andin the binding of Adapter Protein-1 (AP-1) and Adapter Protein-2 (AP-2;Dietrich, et al. (1997) J. Cell Biol. 138:271-281).

XIV. Signal Transduction Via CD94/NKG2C/DAP12 and KIR2DS2/DAP12Complexes

Ligation of KIR2DS2 in transfectants expressing KIR2DS2/DAP12 complexesresults in the tyrosine phosphorylation of DAP12 and other cellularsubstrates and the association of phosphorylated DAP12 with Syk (Lanier(1998) Cell 92:705-707). Ligation of either CD94 or FLAG-DAP12 on Ba/F3transfectants expressing CD94/NKG2C/DAP12 complexes caused tyrosinephosphorylation of numerous cellular proteins, including DAP12 and Syk.These results indicate that cross-linking CD94/NKG2C induces cellularactivation, presumably via DAP12. It was not addressed whether ligationof CD94/NKG2C in the absence of DAP12 or in transfectants expressing theD-A transmembrane FLAG-DAP12 mutant has functional consequences becauseCD94/NKG2C was not efficiently expressed in the absence of wild-typeDAP12.

Unlike CD94/NKG2C, KIR2DS2 molecules are expressed on the cell surfacein the absence of DAP12, although they are unable to induce cellularactivation (Bléry, et al. (1997) J. Biol. Chem. 272:8989-8996; Lanier,et al. (1998) Nature 391:703-707). KIR2DS2+Ba/F3 cells were infectedwith retroviruses encoding either wild-type FLAG-DAP12 or the D-Atransmembrane FLAG-DAP12 mutant receptor. Both KIR2DS2 and the mutantDAP12 protein were expressed on the cell surface. However, the D-Atransmembrane FLAG-DAP12 mutant protein was not co-immunoprecipitatedwith KIR2DS2 from ¹²⁵I labeled transfectants. Furthermore, ligation withanti-KIR mAb failed to activate these cells, whereas directcross-linking of the D-A transmembrane FLAG-DAP12 mutant receptor withanti-FLAG mAb did induce tyrosine phosphorylation of cellular proteins.Like NKG2A and NKG2C, the KIR2DS2 protein has a counterpart, KIR2DL2,that lacks a charged amino acid in the transmembrane and contains anITIM in its cytoplasmic domain. It has been previously reported thatKIR2DL2 is unable to associate with DAP12 (Lanier, et al. (1998) Nature391:703-707). Collectively, these findings indicate that the associationof DAP12 with either KIR2DS2 or CD94/NKG2C complexes likely results frominteractions involving the transmembrane domains of these proteins.

The stoichiometry of DAP12 and KIR2DS2 or CD94/NKG2C in these complexeshas not been determined. A DAP12 disulfide-linked homodimer possessestwo D residues (i.e., one in each DAP12 protein) that could interactwith the K residues present in the transmembranes of KIR2DS2 or NKG2C.Because CD94 lacks charged residues in the transmembrane, DAP12 may beable to function as an adapter permitting the association of two KIR2DS2monomers or two CD94/NKG2C heterodimers with a single DAP12 homodimer.

XV. Association of DAP12 and CD94 in Human NK Cells

CD94/NKG2C receptors previously have been implicated in NK cellactivation (Cantoni, et al. (1998) Eur. J. Immunol. 28:327-338;Houchins, et al. (1997) J. Immunol. 158:3603-3609). A NK cell clone anda polyclonal NK cell line were selected based on their ability tomediate re-directed cytotoxicity against the Fc receptor-bearing P815target cell in the presence of anti-CD94 mAb, suggesting the presence ofan activating CD94-associated receptor complex, probably CD94/NKG2C(Cantoni, et al. (1998) Eur. J. Immunol. 28:327-338). The NK cell cloneand the polyclonal NK cell line were ¹²⁵I labeled, lysed in digitonindetergent to preserve multi-subunit receptor complexes, andDAP12-associated proteins were co-immunoprecipitated using an anti-DAP12antiserum. DAP12-associated proteins were eluted with a pH 11.5 bufferto dissociate the complexes and then the eluted proteins werere-immunoprecipitated with a control mAb or anti-CD94 mAb. For thepolyclonal NK cell line, anti-CD94 mAb specifically reacted with an ¹²⁵Iprotein eluted from the initial anti-DAP12 immunoprecipitate. OnSDS-PAGE analysis, this molecule migrated at ˜70 kD in non-reducingconditions and ˜40 kD in reducing conditions. Equivalent results wereobtained using the NK cell clone. Because CD94 itself does not ¹²⁵Ilabel (Lazetic, et al. (1996) J Immunol. 157:4741-4745; Phillips, et al.(1996) Immunity 5:163-172), it seems likely that the CD94-associated¹²⁵I labeled protein represents NKG2C, although NKG2C-specificserological reagents are not available to confirm this. Nonetheless,these finding demonstrate the existence of a CD94/DAP12 receptor complexon the cell surface of human NK cells.

Paired Activating and Inhibitory Receptors

The KIR gene family encodes receptors that have been implicated ineither cellular activation or inhibition (Biassoni, et al. (1996) J.Exp. Med. 183:645-650; Olcese, et al. (1997) J. Immunol. 158:5083-5086).The inhibitory receptors contain ITIM sequences in their cytoplasmicdomains and lack charged residues in the transmembrane segments, whereasthe activating receptors lack ITIM, often have shorter cytoplasmicregions, and possess a charged amino acid in the transmembrane. Thisgeneral strategy is also evident in the NKG2 (Houchins, et al. (1991) J.Immunol. 158:3603-3609), Ly49 (Smith, et al. (1994) J. Immunol.153:1068-1079), PIR (Hayami, et al. (1997) J. Biol. Chem. 272:7320-7327;Kubagawa, et al. (1997) Proc. Nat'l Acad. Sci. USA 94:5261-5266) and ILT(LIR) (Borges, et al. (1997) J. Immunol. 159:5192-5196; Samaridis andColonna (1997) Eur. J. Immunol. 27:660-665) gene families, which allinclude potentially inhibitory and activating receptors.

It has been shown herein, that DAP12 associates with the activatingisoforms of both the KIR and CD94/NKG2 receptors. The inhibitoryCD94/NKG2A and activating CD94/NKG2C receptors both bind the sameligand, HLA-E (Braud, et al. (1998) J. Immunol. 159:5192-5196). What isthe biological rationale for paired inhibitory and activating receptorsrecognizing MHC class I? The activating CD94/NKG2C/DAP12 receptorcomplex may function to stimulate tyrosine kinases that phosphorylatethe ITIM sequences in the inhibitory NKG2A receptor, resulting in therecruitment of SHP-1 or SHP-2 (Le Drean, et al. (1998) Eur. J. Immunol.28:264-276). However, this seems unlikely since NKG2A and NKG2C aredifferentially expressed within the total NK cell population and only asubset of NK cells expresses both receptors (Cantoni, et al. (1998) Eur.J. Immunol. 28:327-338; and Houchins, et al. (1997) J. Immunol.158:3603-3609). The existence of NK cells expressing CD94/NKG2C, in theabsence of the inhibitory CD94/NKG2A receptor, provides the potentialfor activation of these cells upon encountering HLA-E. HLA-E is broadlyexpressed in normal tissues (Geraghty, et al. (1992) Proc. Natl. Acad.Sci. USA 89:2669-2673; Lee, et al. (1998) Proc. Natl. Acad. Sci. USA95:5199-5204; Ulbrecht, et al. (1992) J. Immunol. 149:2945-2953);therefore, activation of NK cells via CD94/NKG2C/DAP12 might result inautoimmunity. However, recent studies suggest that all NK cell clonesappear to express at least one inhibitory receptor (either a KIR orCD94/NKG2A) against a self MHC class I ligand, thus preventingdestruction of normal autologous tissues (Uhrberg, et al. (1997)Immunity 7:753-763; Valiante, et al. (1997) Immunity 7:739-751). NK cellclones expressing activating CD94/NKG2C/DAP12 receptors and aninhibitory KIR against a self class I ligand could potentially recognizeand eliminate host cells that have lost expression of the KIR class Iligand, but retained expression of HLA-E. This model requiresexperimental testing, but would provide defense against pathogens thatencode leader peptides competent to bind HLA-E, but down-regulateexpression of conventional MHC class I molecules as a consequence ofinfection.

Transfectants

cDNA used were human CD94 (Chang, et al., 1995), NKG2A and NKG2C(Houchins, et al. (1991) J. Immunol. 158:3603-3609), KIR2DS2 (NKAT5,(Colonna and Samaridis, 1995)) and FLAG-DAP12 (Lanier, et al. (1998)Nature 391:703-707). The D-A transmembrane FLAG-DAP12 mutant cDNA withan A residue (codon GCC) substituted for the D residue (codon GAC) andthe K-L transmembrane NKG2C mutant cDNA with a L residue (TTA)substituted for K (codon AAA) were generated by PCR mutagenesis usingconventional techniques. A NKG2C cDNA containing a FLAG epitope on theCOOH terminus immediately prior to the NKG2C stop codon was generated byPCR. cDNA were sequenced and subcloned into the pMX-neo or pMX-puroretroviral vectors (Onihsi, et al. (1996) Exp. Hematology 24:324-329).Plasmid DNA was transfected into Φ-NX-E ecotropic retrovirus packagingcells (a generous gift from G. Nolan (Stanford University)) usinglipofectamine (Gibco-BRL) (Onihsi, et al., 1996). Viral supernatantswere collected two days later and used to infect mouse Ba/F3 pre-B cells(Onihsi, et al., 1996). Two days post-infection cells were switched toselection medium and Ba/F3 cells stably expressing human NK cellreceptors were sorted by flow cytometry for homogeneous high levelexpression.

Antibodies and Flow Cytometry

mAbs used were anti-CD94 (DX22; Phillips, et al. (1996) Immunity5:163-172) or HP-3D9 mAb (Lopez-Botet (1995), pp. 1437-1439, inSchlossman, et al. (eds.) Leucocyte Typing V. Oxford University Press,Oxford; anti-KIR2D mAb (DX27; Phillips, et al. (1996) Immunity5:163-172), anti-NKR-P1A (DX1; Lanier et al. (1994) J. Immunol.153:2417-2428), anti-FLAG (M2 mAb, Kodak), anti-NKG2A/C (8E4 mAb;Houchins, et al. (1997) J. Immunol. 158:3603-3609) and control mouseIgG1 mAb (Becton Dickinson, San Jose, Calif.). Rabbit antiserum specificfor the CD94/NKG2A and CD94/NKG2C heterodimers was prepared as described(Lazetic, et al. (1996) J Immunol. 157:4741-4745). FITC conjugated goatanti-rabbit Ig and FITC conjugated anti-mouse Ig second antibodies werepurchased from CalTag (So. San Francisco, Calif.). Immunofluorescenceand flow cytometry were performed as described (Lanier and Recktenwald(1991) Methods: A Companion to Methods in Enzymology 2:192-199).

Biochemistry

Transfected Ba/F3 cells were labeled with ¹²⁵I and solubilized indigitonin lysis buffer (pH 7.8, 1% digitonin, 0.12% Triton-X100, 150 mMNaCl, 20 mM triethanolamine, 0.01% NaN₃, and protease inhibitors;Lanier, et al. (1989) Nature 342:803-805). Cell lysates were incubatedon ice for 2 hr with Pansorbin (Calbiochem) coated with rabbitanti-mouse/rat Ig (Sigma) and anti-CD94 (DX22 mAb), anti-FLAG (M2 mAb)or control IgG and then washed. Immunoprecipitates were resuspended inSDS-PAGE sample buffer in the presence or absence of 10%2-mercaptoethanol, run on 18% Tris/glycine gels (Novex) and visualizedby using a PhosphorImager (Molecular Dynamics).

A human NK cell clone and a polyclonal human NK cell line (CD3−,CD56+peripheral blood NK cells cultured as described (Yssel, et al. (1984) J.Exp. Med. 160:239-254) were labeled with ¹²⁵I and solubilized indigitonin lysis buffer. ¹²⁵I cell lysates were pre-cleared overnightwith Pansorbin coated with rabbit Ig and then incubated on ice for 2 hrwith Pansorbin coated with an affinity purified rabbit anti-DAP12antiserum (generated by standard methods against a GST fusion proteincontaining the entire cytoplasmic domain of human DAP12).DAP12-associated proteins were eluted in 25 μl 50 mM diethylamine (pH11.5) and transferred to 0.5 ml 1% NP-40 lysis buffer (50 mM Tris, 150mM NaCl, pH 8.0 containing protease inhibitors) with 10 mg/ml BSAcarrier protein. The DAP12-associated eluted proteins werere-immunoprecipitated anti-CD94 mAb (HP-3D9 and DX22) coupled Sepharosebeads or anti-NKR-P1A mAb (DX1) coupled Sepharose beads (used as anegative control). Immunoprecipitates were washed in 1% NP-40 lysisbuffer, resuspended in SDS-PAGE sample buffer in the presence or absenceof 10% 2-mercaptoethanol, run on 18% Tris/glycine gels and visualized byusing a PhosphorImager.

Western blot analysis using anti-FLAG (M2 mAb) or anti-NKG2A/C (8E4 mAb;Houchins, et al. (1997) J. Immunol. 158:3603-3609) was performed asdescribed in Phillips, et al. (1996) Immunity 5:163-172. 8E4 mAb detectsboth NKG2A and NKG2C by Western blot analysis, but does notimmunoprecipitate or bind to these antigens in immunofluorescenceassays.

Cellular Stimulation

Transfected Ba/F3 cells were suspended in cold PBS with 0.5% BSA at5×10⁷ cells/ml containing 20 μg/ml mAb recognizing CD94, FLAG-DAP12, orKIR2DS2. Cells were incubated on ice for 30 minutes, washed, resuspendedin 10 μg/ml goat anti-mouse IgG F(ab′)₂ (Jackson Immunoresearch), andincubated for three minutes at 37° C. Cells were pelleted, resuspendedat 10⁸/ml in ice cold lysis buffer (1% NP-40, 10 mM Tris, pH 7.4, 150 mMNaCl containing the protease and phosphates inhibitors-aprotinin,leupeptin, PMSF, EDTA, NaVO₄, and NaF) as described (Lanier, et al.(1998) Nature 391:703-707). Syk and FLAG-DAP12 were immunoprecipitatedwith rabbit anti-Syk antiserum (generously provided by Joe Bolen, DNAX)or anti-FLAG (M2 mAb). Cell lysates (2−3×10⁶ cell equivalents) andimmunoprecipitates were run on Tris/glycine gels, blotted onto Immobilonmembranes (Millipore), blocked, probed with horseradishperoxidase-conjugated anti-phosphotyrosine mAb 4G10 (UpstateBiotechnology), washed, and developed with a chemiluminescent substrate(Pierce).

XVI. Murine DAP12 Associates with Ly49D or Ly49H

Several members of the Ly49 receptor family inhibit NK cell-mediatedlysis of targets expressing appropriate MHC class I molecules. Ly49D andLy49H, two Ly49 molecules that lack Immunoreceptor Tyrosine-basedInhibitory Motifs (ITIM) in their cytoplasmic domains, associate withmouse DAP12, a molecule which possesses an Immunoreceptor Tyrosine-basedActivation Motif (ITIM). Co-transfection of either Ly49D or Ly49H withDAP12 induces surface expression of both Ly49 and DAP12. The Ly49/DAP12complex was co-immunoprecipitated from the transfected cells,demonstrating a physical association of DAP12 with Ly49D or Ly49H in theplasma membrane. Stimulation of transfectants with antibodiesrecognizing either Ly49D or Ly49H results in cellular activation asassessed by induction of tyrosine phosphorylation of multiple cellularsubstrates.

NK cells express receptors for MHC class I which upon recognition ofappropriate polymorphic class I ligands deliver an inhibitory signal,resulting in the inhibition of target lysis. Mouse Ly49A, the prototypicinhibitory receptor for H-2 (Karlhofer, et al. (1992) Nature 358:66), isa homodimeric type II integral membrane protein of the C-type lectinfamily expressed on natural killer cells and a small population of Tcells. The Ly49 family includes 9 genes, Ly49A through I (Smith, et al.(1994) J. Immunol. 153:1068; Brennan, et al. (1994) J. Exp. Med.180:2287; Takei, et al. (1997) Immunol. Rev. 155:67). Seven of the Ly49molecules (Ryan and Seaman (1997) Immunol Rev. 155:79) possess an ITIM(V/IxYxxL/V) (Thomas (1995) J. Exp. Med. 181:1953; Lanier (1997)Immunity 6:371) in their cytoplasmic domains. The phosphorylated ITIM inLy49A and Ly49G2 bind the cytoplasmic tyrosine phosphatases SHP-1 andSHP-2 (Olcese, et al. (1996) J. Immunol. 156:4531; Nakamura, et al.(1997) J. Exp. Med. 185:673; and Mason, et al. (1997) J. Immunol.159:4187). Engagement of Ly49A by its ligand H-2D^(d) interrupts earlyactivation events induced by interaction of NK cells with target cells(Nakamura, et al. (1997) J. Exp. Med. 185:673). Ly49D and Ly49H, lackITIM and possess a positively charged arginine residue within theirtransmembrane domains. Ly49D is unable to deliver an inhibitory signaland in fact may activate NK cells (Mason, et al. (1996) J. Exp. Med.184:2119).

Human NK cells express a functionally analogous set of molecules, thekiller cell inhibitory receptors (KIR), which belong to theimmunoglobulin superfamily (Lanier (1997) Immunity 6:371). KIR, likeLy49, can be divided into two sub-families based on the presence orabsence of ITIM in their cytoplasmic domains. KIR2DL or KIR3DL possessITIM and inhibit lysis of targets expressing their MHC class I ligands.KIR isoforms lacking ITIM (KIR2DS) possess a positively charged residuein their transmembrane domains and deliver an activating signal(Moretta, et al. (1995) J. Exp. Med. 182:875; Biassoni, et al. (1996) J.Exp. Med. 183:645). DAP12, which non-covalently associates with KIR2DS2(Lanier, et al. (1998) Nature 391:703-707), possesses an ITAM in itscytoplasmic tail and a negatively charged aspartic acid residue in itstransmembrane domain. Ligation of the KIR2DS2/DAP12 complex results incellular activation. The association of mouse DAP12 with Ly49D andLy49H, and the ability of these complexes to activate downstreamsignaling pathways was examined.

Transcripts of Ly49D and Ly49H are present in IL-2 activated NK cells(20). Ly49D is expressed on ˜50% of NK cells (Mason, et al. (1996) J.Exp. Med. 184:2119), and is associated with a tyrosine phosphoprotein of16 kD (Mason, et al. (1998) J. Immunol. 160:4148-4152). Murine NK cells,like human NK cells, transcribe mRNA for DAP12, a molecule whichassociates with the activating KIR2DS and mediates cellular activation(Lanier, et al. (1998) Nature 391:703). To examine if Ly49D or Ly49Hassociated with DAP12, Ba/F3 cells were stably transfected with anepitope-tagged mouse DAP12 (DAP12-FLAG). Ba/F3-DAP12-FLAG cells do notexpress DAP12 on the cell surface. Ba/F3 or the Ba/F3-DAP12transfectants were then infected with retroviruses encoding eitherLy49D, a myc epitope tagged Ly49H (Ly49H-myc), or as a control Ly49A.Neither Ly49D nor Ly49H-myc was expressed at appreciable levels on thecell surface when transfected into Ba/F3 cells. In contrast,transfection of Ba/F3-DAP12-FLAG cells with either Ly49D or Ly49H-mycresulted in high level surface expression of both Ly49 and DAP12-FLAG,suggesting that Ly49D and Ly49H associate with DAP12.

It was examined whether the charged residues in the transmembranes ofLy49 and DAP12 are important for their association. Ly49A shares 86%amino acid identity with Ly49D in its extracellular domain, but lacksthe arginine in its transmembrane segment. In contrast to Ly49D orLy49H, when Ly49A was stably transfected into Ba/F3 or Ba/F3-DAP12-FLAGcells, it was expressed at the cell surface alone or in the presence ofDAP12-FLAG and failed to induce surface expression of DAP12-FLAG.Interactions between Ly49D or Ly49H-myc and DAP12 are notspecies-restricted because both Ly49 molecules were expressed on thesurface of Ba/F3-human DAP12-FLAG transfectants. However, neither Ly49Dor Ly49H were expressed on the surface of Ba/F3 cells stably transfectedwith a mutant human DAP12 molecule in which the negatively chargedaspartic acid in the transmembrane was mutated to leucine. Therefore,both Ly49D and Ly49H must associate with DAP12 to effectively reach thecell surface and their interaction is likely mediated by the oppositelycharged residues in the transmembranes of DAP12 and Ly49.

To confirm that Ly49D and Ly49H non-covalently associate with DAP12 atthe cell surface, Ly49D/DAP12-FLAG or Ly49H-myc/DAP12-FLAG Ba/F3transfectants were surface iodinated, lysed with digitonin, andimmunoprecipitates were analyzed by SDS-PAGE. Immunoprecipitation ofBa/F3-Ly49D/DAP12-FLAG lysates with anti-Ly49D showed two iodinatedspecies with sizes consistent with their identity as Ly49D andDAP12-FLAG. An identical pattern was observed with anti-FLAG, confirmingthat the two species are Ly49D and DAP12-FLAG. Immunoprecipitation ofBa/F3-Ly49H-myc/DAP12-FLAG lysates with anti-myc or anti-FLAG showed asimilar pattern. These results demonstrate a physical interaction ofLy49D or Ly49H with DAP12 in the plasma membrane.

XVII. Ly49/DAP12 Complexes Transmit Intracellular Activating Signals

Since DAP12 possesses an ITAM and engagement of Ly49D activates NK cells(Mason, et al. (1996) J. Exp. Med. 184:2119), it was asked if theLy49/DAP12 complexes transmit an activating signal. Crosslinking ofLy49D/DAP12-FLAG and Ly49H-myc/DAP12-FLAG transfectants with anti-Ly49or anti-FLAG resulted in tyrosine phosphorylation of many cellularproteins including DAP12-FLAG and Syk in both cell lines. These dataprovide evidence that Ly49D/DAP12 and Ly49H/DAP12 form functionalcomplexes at the cell surface which upon ligation can initiate cellularactivation.

What are the physiological ligands for these activating receptors? Ly49Dshares 86% amino acid identity in its extracellular domain with Ly49A(Smith, et al. (1994) J. Immunol. 153:1068), an inhibitory receptor thatbinds H-2D^(d) and H-2D^(k) (Brennan, et al. (1996) J. Exp. Med.183:1553; Kane (1994) J. Exp. Med. 179:1011; Daniels, et al. (1994) J.Exp. Med. 180:687). Ly49H shares 90% amino acid identity in itsextracellular domain with another inhibitory receptor Ly49C (Brennan, etal. (1994) J. Exp. Med. 180:2287), which interacts with several class Imolecules, including H-2K^(b) (Brennan, et al. (1996) J. Exp. Med.183:1553). Thus, these activating forms of Ly49 may interact with MHCclass I molecules. Evidence for positive allorecognition by NK cellsboth in vivo and in vitro exists in the rat (reviewed in Rolstad, et al.(1997) Immunol. Rev. 155:91). Similarly, mouse NK cells recognizeallogeneic bone marrow cells expressing certain class I molecules in apositive fashion and mediate their rejection in vivo (Ohlen, et al.(1989) Science 246:666; George, et al. (1997) Immunol Rev. 155:29). Ithas been shown that mouse Ly49D and Ly49H associate with DAP12 and formactivating receptors, providing a possible explanation for positiveallorecognition by NK cells.

How can the existence of both activating and inhibitory NK receptorswhich recognize class I ligands be reconciled? Three models areenvisioned. In the first model, engagement of activating receptors wouldfunction during development to promote maturation of immature NK cells.However, so far there is no evidence for the appearance of activatingreceptors prior to inhibitory receptors during development. A secondmodel proposes that an NK cell possesses activating and inhibitoryreceptors for the same class I ligand. Upon engaging class I, theactivating receptor would recruit a protein tyrosine kinase thatphosphorylates the ITIM of the inhibitory receptor, resulting in NK cellinactivation. While most human NK cell clones possess at least oneactivating and one inhibitory receptor they do not necessarily possess apair capable of recognizing the same ligand. Finally, a third modelpredicts that NK cells express inhibitory and activating receptor fordifferent class I alleles. In this model, engagement of the inhibitoryreceptor dominates if ligands for both receptors are engaged. If theligand for the inhibitory receptor is down-regulated or lost, theactivating receptor could trigger lysis of the “abnormal” cell if itsligand is present. This model has the advantage that multiple inhibitoryand activating receptors could be expressed by the same cell, aprediction more in line with the findings in NK clones. Yet, in the caseof loss of all MHC class I molecules by a target cell, other activatingmechanisms would have to initiate lysis by the NK cell.

XVIII. Isolation of Associated Proteins

DAP12 remains localized intracellularly when expressed in cells in theabsence of associating partners. This observation was exploited with thepurpose of cloning novel DAP12-associating proteins, e.g., to expressionclone genes necessary in the process of cellular localization to themembrane. Cells lacking the associated proteins were transfected withthe DAP12, and the protein remained intracellularly localized. Thesecells could be used to expression clone necessary accessory proteins forDAP12 surface localization. The strategy had been labeled “DAP-trap”.

To this end, a FLAG-tagged form of mouse DAP12 was expressed in 293Tcells using an expression vector, e.g., pREP10. In the presence ofhygromycin, a stable DAP12 expressing cell line was selected, DT381. Toreduce the background of spontaneous DAP12 expression at the cellsurface, DT381 cells were negatively selected by flow cytometry usingthe M2 anti-FLAG mAb (Kodak). To clone novel DAP12 associating proteins,a J774 macrophage cell line derived pJEF14 expression library wastransfected into DT381 cells. Forty-eight hours after transfection, thecells were selected for cell surface expression of DAP12 by flowcytometry. This was performed by two color staining: DAP12 wasvisualized using the M2 anti-FLAG mAb, followed by a biotin-conjugatedanti-mouse IgG1 mAb (#02232D Pharmingen), followed by a streptavidin-PEthird step incubation. Fc receptors on transfected DT381 cells werevisualized using the directly FITC-conjugated anti-CD16/32 mAb 2.4G2(#01244D Pharmingen). Only single PE positive cells were sorted.Staining with the anti-CD16/32 mAb was necessary to avoid the cloning ofFc receptors which are abundantly present in J774 cells.

The plasmids from the sorted cells were rescued and the DNA wasretransformed into DH10B bacteria. Sublibraries were obtained andsubjected to a novel round of expression cloning. After three rounds ofselection, 500 single bacterial colonies from the third sublibrary weregrown in a 96 well plate format to construct a three dimensional matrixof consisting of 5×12×8 colonies. DNA obtained from pools of each X, Y,and Z coordinate of this matrix was again transfected into DT381 cellsand the transfectants were screened for DAP12 surface expression.

This resulted in the identification of two identical clones, bothencoding a 165 amino acid type II transmembrane protein of the C-typelectin superfamily. This gene/protein was designated Myeloid DAP12associating Lectin-1 (MDL-1). This embodiment of MDL-1 from the mousehas an intracellular region of 2 residues, a transmembrane region of 23residues, and a 140 residue extracellular region containing the C-typelectin domain. The transmembrane segment possesses a charged amino acid,an the extracellular region has three putative N-glycosylation sites.BLAST searching revealed a highly homologous full length mouse EST,AA186015, which was identical to the two above mentioned clones, withthe exception that this clone has an extra stretch of 75 nucleotidesresulting in a 25 residue additional stretch extracellularly justoutside of the transmembrane region. Thus, there exist two embodiments,a short form and long form. The rest of the sequences are identical.

Searching within a DNAX sequence database revealed a homologous humanEST, #97-1128A12, which encodes a human homologue of MDL-1. The mouseMDL-1 appears to be encoded by a single gene, in contrast to manyrelated surface proteins, which may occur in families of genes. Themouse MDL-1 expression is restricted to monocytes, macrophages anddendritic cells.

Because the MDL-1 gene appears to be crucial in localization of theDAP12 to the membrane, and possesses interesting structural features, itis likely that the MDL-1 associates with the DAP12 in a membranecomplex. Thus, disruption of the complex may lead ton interestingblocking of function of the DAP12-receptor complex. This suggestsobvious approaches to small molecule drug screening for compounds whichwould interfere with association. Alternatively, transmembrane fragmentsmay block functional association. Antibodies to the extracellularregions, either of proteins alone, or the combination of components inthe functional complexes, would be useful in diagnostic or therapeuticcontexts.

DAP10 also seems to associate with an accessory protein. In particular,immunoprecipitation of DAP10 under mildly denaturing conditions resultsin co-immunoprecipitation of a protein band of about 40-41 kD.Neuraminidase treatment, or O-glycanase treatment, result in a decreasein molecular weight to about 38-39 kD. N-glycanase treatment causes adecrease in molecular weight to about 28-30 kD. These suggest that theprotein is about 26-30 kD without glycosylation. Standard ormicrosequencing methods can be applied to protein isolated byimmunoprecipitation. With sequence, redundant PCR primers, or othertechniques can be applied to isolate the gene. Alternatively, sequencemay allow identification of the gene by matches in sequence databases.

Moreover, the DAP10 is also subject to the DAP-trap strategy. Expressioncloning techniques can be applied, as with the DAP12, to clone the genefrom a cDNA library. Distribution information will allow selection ofthe appropriate cell lines and cDNA libraries for such.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited bythe terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An isolated or purified nucleic acid encoding a polypeptidecomprising the sequence of SEQ ID NO
 2. 2. An expression vectorcomprising the nucleic acid of claim
 1. 3. A host cell comprising theexpression vector of claim
 2. 4. The host cell of claim 3, wherein thehost cell is: a) a prokaryotic cell; b) a eukaryotic cell; c) an insectcell; or d) a yeast cell.
 5. A method of producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:2 comprising: a)culturing the host cell of claim 4 under conditions suitable forexpression of the polypeptide; and b) isolating the polypeptide.
 6. Thenucleic acid of claim 1, wherein the nucleic acid comprises SEQ ID NO:1.7. The nucleic acid of claim 6, wherein the nucleic acid is detectablylabeled.
 8. A method of making a nucleic acid duplex comprisingcontacting the nucleic acid of claim 1 with a complementary nucleic acidunder conditions suitable for the formation of the duplex and detectingthe duplex.